T cell response magnitudes increase with increasing antigenic dosage. However, it is unclear whether ligand density only modulates the proportions of responding ligand-specific T cells or also alters responses at the single cell level. Using brief (3 h) exposure of TCR-transgenic mouse CD8 T cells in vitro to varying densities of cognate peptide-MHC ligand followed by ligand-free culture in IL-2, we found that ligand density determined the frequencies of responding cells but not the expression levels of the early activation marker molecule, CD69. Cells with low glucose uptake capacity and low protein synthesis rates were less ligand-sensitive, implicating metabolic competence in the response heterogeneity of CD8 T cell populations. Although most responding cells proliferated, ligand density was associated with time of entry into proliferation and with the extent of cell surface TCR downmodulation. TCR internalization was associated, regardless of the ligand density, with rapidity of c-myc induction, loss of the cell cycle inhibitor p27kip1, metabolic reprogramming, and cell cycle entry. A low affinity peptide ligand behaved, regardless of ligand density, like a low density, high affinity ligand in all these parameters. Inhibition of signaling after ligand exposure selectively delayed proliferation in cells with internalized TCRs. Finally, internalized TCRs continued to signal and genetic modification of TCR internalization and trafficking altered the duration of signaling in a T cell hybridoma. Together, our findings indicate that heterogeneity among responding CD8 T cell populations in their ability to respond to TCR-mediated stimulation and internalize TCRs mediates detection of ligand density or affinity, contributing to graded response magnitudes.

CD8 T cells are capable of identifying specific target peptide-MHC class I (pMHCI) complexes present on the APC surface with great sensitivity and discrimination amid other noncognate pMHCI species (1). The TCR–pMHCI interaction leads to a complex series of signaling events resulting in activation, proliferation, and differentiation of CD8 T cells into effector cytotoxic cells (2). The magnitude of CD8 T cell responses, both in vivo and in vitro, is known to increase with ligand dose (36), as well as with ligand affinity for the TCR (79). However, it is not yet clear how response-capable CD8 T cell populations convert detection of these graded ligand densities into decisions controlling the magnitude of the corresponding responses. Do more and more response-capable CD8 T cells actually become recruited, and/or do recruited individual CD8 T cells respond to a greater extent, perhaps by greater proliferation?

Although CD8 T cell responses have high specificity, they also have high sensitivity, creating the expectation that, once they actually cross the threshold of response, they generate a strong response relatively independent of ligand density (1, 1012). In fact, models of T cell activation posit serial TCR triggering by a single ligand pMHCI as a signal amplifying mechanism to provide specificity and sensitivity (10).

Analysis of such issues is complicated by a number of features of T cell biology. In natural polyclonal T cell populations, the subset of cells that can respond to a given pMHC complex is heterogeneous in its affinity for the ligand. Even in a TCR-transgenic monoclonal T cell population, strength of TCR signaling is affected by not simply the ligand density and affinity but also the duration of ligand availability (13, 14), which could potentially allow multiple interactions to occur between responding T cells and ligand-bearing APCs, especially in vivo, as could be the case with previous studies showing that increasing ligand doses lead to greater degrees of CD8 T cell proliferation (36, 1517).

The issue of duration of T cell–APC interactions is complicated by findings that responding T cells build quasi-stable interfaces, or immunological synapses, with ligand-bearing APCs (18) and that TCRs are internalized from these interfaces (19). Although the precise location, kinetics, and functional significance of TCR internalization are still unclear, synapse formation and TCR internalization appear to be correlates of successful T cell activation (20, 21). There is also evidence that responding T cells may remove ligand from APC surfaces (22, 23), although, once again, the relationship of this phenomenon with TCR internalization and its significance for successful T cell activation is unclear.

The issue of how ligand density and affinity determine T cell responses is also complicated by a number of other features. Firstly, naturally generated pMHCI complexes, loaded with proteasomally derived peptides, do not appear to be uniformly distributed on the APC surface but as clusters (2427). Further, specific but noncognate self-pMHCI complexes on the same APC may also participate during cognate ligand recognition by T cells (2831). The relationship between ligand density and ligand affinity is also complex; at certain ligand densities, very high ligand affinity is counter-productive for successful CD8 T cell activation, perhaps due to failure of serial TCR triggering (32), whereas at other ligand densities, T cell activation increases as a function of ligand affinity (33).

On this background, we have used a simplified system of CD8 T cell activation in vitro using a defined duration of contact between TCR-transgenic monoclonal CD8 T cells and APCs pulsed with varying concentrations of synthetic peptides generating high or low affinity TCR ligands, followed by APC-independent culture and analysis of T cells activated during contact. We find that, although populations of monoclonal CD8 T cells show graded frequencies of response to ligand density and affinity, cells that have been successful in crossing the threshold of response during T cell–APC contact respond well regardless of ligand density or affinity, with subtle modulation of the time for cell cycle entry by density- or affinity-dependent TCR internalization decisions. Our data suggest that understanding the basis of the heterogeneity in the ability to respond to TCR-mediated stimulation within ostensibly homogenous T cell populations is significant for our understanding of T cell immune responses.

C57BL/6-Tg(TcraTcrb)1100Mjb/J TCR transgenic OT1 mice specific for the H-2Kb–restricted OVA1 peptide SIINFEKL, and C57BL/6-Tg(UBC-GFP)30Scha/J (B6.GFP) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and maintained in the animal facility of the National Institute of Immunology. B6.GFP mice were bred with OT1 mice to obtain OT1.GFP mice for some experiments. All mice were maintained and used according to the relevant rules and regulations of the Government of India and with the approval of the Institutional Animal Ethics Committee.

The epithelial cell line PS-SW expressing H-2Kb (gift of Prof. C. Janeway, Yale Medical School, New Haven, CT) was used to present cognate peptide in vitro to OT1 T cells. B3Z is a T cell hybridoma that uses the OT1 TCR to recognize the OVA1 peptide on H-2Kb (34). Both cell lines were maintained in RPMI 1640 medium with 10% FBS at 37°C in 5% CO2.

Bone marrow–derived dendritic cells (BMDCs) were cultured as described (35). Briefly, BMDCs were grown by culturing mouse bone marrow cells with recombinant granulocyte monocyte CSF (rGM-CSF; PeproTech, Rocky Hill, NJ) for 7 d, with periodic growth factor replenishment. Semiadherent cells obtained on day 7 were used for experiments.

Abs for mouse CD8, CD69, TCRVα2, CD25, CD62L, CD44, and TCR-CD3, phospho-ZAP70, LAMP-1, and p27kip1 (from BD Biosciences, San Jose, CA), and for IFN-γ and phospho-S6 (pS6) (from eBioscience, San Diego, CA) were used. F(ab′)2 fragments of goat anti-mouse IgG1 coupled to Alexa Fluor 647 (Jackson ImmunoResearch Laboratories, West Grove, PA) were used where appropriate. Recombinant IL-2 (Roche, Basel, Switzerland), CFSE, Cell Trace Violet (CTV), Sytox Red, Mitotracker Green, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), and Click-iT kits for 5-ethynyl-2′-deoxyuridine (EdU) and l-homopropargylglycine (HPG) (from Molecular Probes, Carlsbad, CA) were used. Commercially synthesized SIINFEKL (Peptron, Daejeon, South Korea) and SIIQFEKL (Sigma-Aldrich, St Louis, MO) were used as indicated. The PI3K inhibitor LY294002 (Calbiochem, San Diego, CA) was used where indicated. Cells were cultured in RPMI 1640 medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% FBS (Sigma-Aldrich), 2 mM l-glutamine and antibiotics (Sigma-Aldrich).

PS-SW cells grown as monolayers or BMDCs in six-well plates were used as APCs. The monolayer was washed with media and incubated with various peptide concentrations as indicated for 6 h, followed by three washes to remove unbound peptides. CFSE- or CTV-labeled OT1 cells purified from spleens and lymph nodes of OT1 mice were added onto the APC monolayer. After brief stimulation of 3 h at 37°C, the lymphocyte suspension was removed gently without disturbing the APC monolayer, and stained for flow cytometry and electronic sort purification. Sort-purified OT1 CD8+ CD69+ T cells were cultured in the presence of 10 U/ml IL-2. As a control, cells were incubated with unpulsed APCs and cultured similarly. In some experiments, sort-purified cells were treated with the PI3K inhibitor LY294002 (Calbiochem) for 4 h, then washed and cultured further. Restimulation of primed OT1 T cells was performed where indicated using 50 ng/ml PMA and 125 ng/ml ionomycin (Sigma-Aldrich) for 5 h, followed by intracellular staining for IFN-γ.

Cells were incubated with staining Abs on ice for 30 min. Control samples were incubated in staining buffer alone (PBS containing 1% FCS and 0.1% sodium azide) or with an appropriate isotype-matched control Ab. The cells were then washed with PBS. For detecting intracellular proteins, cells were fixed with BD Cytofix buffer (BD Biosciences) for 30 min on ice and permeabilized with BD PhosphoflowPerm Buffer II (BD Biosciences) for 30 min on ice, followed by staining. Dead cells were excluded using Sytox dye staining. Samples were analyzed on FACSVerse or FACSARIA III (BD Biosciences) and data analyzed with FlowJo software (Treestar, Ashland, OR). Naive CD8 T cells were sort purified as CD8+CD62LhiCD44lo.

Glucose uptake was determined by flow cytometry with a fluorescent deoxyglucose analog 2-NBDG (Molecular Probes). Cells were incubated in glucose-free RPMI 1640 medium containing 100 μM 2-NBDG for 30 min at 37°C and then washed twice with cold PBS. For estimating mitochondrial mass, cells were incubated in 50 nM Mitotracker Green (Molecular Probes) in the dark for 30 min at 37°C.

EdU is a nucleoside analog of thymidine that can be incorporated into DNA during active DNA synthesis, whereas HPG is an amino acid analog that can be incorporated into proteins during active protein synthesis. Detection of both incorporated analogs can be achieved with click chemistry-based copper-catalyzed azide-alkyne reactions mediating covalent binding of a fluorochrome to the analogs. For DNA synthesis detection, sort-purified cells were pulsed with 10 μM EdU for 2 h at indicated time points. For protein synthesis detection, cells were washed with PBS, followed by incubation in methionine-free RPMI 1640 medium for 1 h. Cells were then pulsed with 50 μM HPG in methionine-free RPMI 1640 medium for 2 h. For detecting both incorporated analogs, cells were fixed with 4% paraformaldehyde at room temperature for 10 min, then permeabilized with 0.2% Triton X-100 at 4°C for 30 min, and were then incubated for 30 min at 37°C in staining solution containing 20 μM Alexa Fluor 488–azide according to the manufacturer’s protocols (Invitrogen).

After activation, incubation, and purification as indicated, OT1 cells were washed with PBS and lysed on ice in lysis buffer (1% Triton X-100, 50 mM Tris pH 7.4, 0.5 M EDTA, 150 mM NaCl, 0.1% SDS) supplemented with a protease inhibitor mixture (Sigma) for 30 min. Then, 50 μg of protein was run on SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked with 2% BSA and probed with anti-p27kip1 (clone 57/kip1/p27; BD Biosciences) and an appropriate HRP-labeled anti-mouse Ig (Jackson Immunoresearch). Membranes were washed and probed with anti–β-actin (Santa Cruz, Dallas, TX) for normalizing inputs.

B3Z cells were transfected with a dominant-negative (DN) mutant of Rab7 (DN-Rab7) (36) by electroporation followed by G418 selection. Control or DN-Rab7–expressing B3Z cells were stimulated for 3 h with APCs pulsed with N4 or Q4 peptides as indicated and then removed from APC monolayers followed by culture in APC-free medium. The induction of IL-2 promoter-driven β-galactosidase (bGal) production was assayed 10 h later by a colorimetric assay as previously described (34) by incubating cells with substrate buffer (0.15 M chlorophenol red-β-d-galactoside in 100 mM mercaptoethanol, 0.125% NP-40 and 9 mM MgCl2) for 8 h and measuring absorbance at 570 nm.

For detailed flow cytometry and microscopy experiments, B3Z cells were transiently transfected with wild-type (WT), DN or dominant-active (DA) versions of genes coding for various regulators of endocytic trafficking in CMV promoter-driven expression vectors containing a GFP gene tag. The gene interventions used were: the C-terminal fragment of the clathrin-associated protein AP180 (AP180C), WT and DN versions of the clathrin-associated protein Eps15 (WT-Eps15, DN-Eps15), or of dynamin-2 (WT-dyn, DN-dyn), or of the small GTPases Rab7 (WT-Rab7, DN-Rab7), Rab11 (WT-Rab11, DN-Rab11), or Rab21 (WT-Rab21, DN-Rab21), or WT, DA, or DN versions of Rab5 (WT-Rab5, DA-Rab5, DN-Rab5) or Rab22 (WT-Rab22, DA-Rab22, DN-Rab22) (kind gifts from Dr. S. Mayor, National Center for Biological Sciences, Bangalore, India). Fugene6 (Roche) was used for transfection according to the manufacturer’s protocols using 10 μg plasmid DNA per million cells. After 24 h, transfected B3Z cells were stimulated for 2–3 h with PS-SW APCs pulsed with various concentrations of SIINFEKL, then removed from APC monolayers followed by culture in APC-free medium. The induction of bGal expression was assayed at various time points later by fixing and permeabilizing the cells as described and staining them with a PE-conjugated anti-bGal Ab (Prozyme, Hayward, CA) followed by flow cytometric analysis.

B3Z cells activated on SIINFEKL-pulsed APCs were harvested at various time points, fixed (4% paraformaldehyde, 20 min, 37°C), permeabilized (0.03% saponin in PBS, 20 min, ambient temperature), and blocked with BSA in PBS prior to incubation with Abs in the same medium. Rabbit and mouse primary Abs were detected using fluorophore-labeled Fc-specific anti-rabbit IgG or anti-mouse IgG.

Confocal imaging was performed on a FluoView FV1000 confocal microscope (Olympus) with factory set dichroics and Argon-Krypton lasers using the FluoView software with a step size of 0.1 μm and a pixel size of 0.15 μm, to acquire 8-bit images with a 100× (1.45 NA) objective. Colocalization between internalized vesicles and endocytic markers was quantified using two software packages, MultiSpots and MultiColloc (36), variations of custom-developed software, Spots and Coloc as described previously (37). The images were corrected for background fluorescence using the produce background correction image procedure in MetaMorph image analysis software using parameters suitable for producing a local background image and at the same time preserving the intensity of the endosomes. The background corrected images were then processed through MultiSpots to identify endosomes using parameters defined by pixels above a threshold (set by inspection for each image) and area (minimum = 3 pixel, maximum = no upper limit). A trimming procedure was applied to isolate and segregate individual endosomes based on the inclusion of pixels with intensities >0.3 of maximal intensity within a unit of connected pixels having intensity greater than the set threshold. This was achieved by an iterative procedure using a step size (0.01) for each iteration, which limits for the number of iterations of the trimming procedure. The resultant image contained a set of identified spots or endosomes quantified in terms of net intensity per spot and total pixel area per spot. The two spotted images of different fluorophore-labeled markers were matched up against each other to identify the endosomes where the two molecules were colocalized. Endosomes in a tracer image were compared with endosomes in a reference image using MultiColloc, and endosomes with 50% or more area overlap were considered to be colocalized. The resultant image displays all the spots that are colocalized using a particular tracer-reference image pair. Cell outlines were drawn based on the phase contrast image of the cell, and the net intensity of each fluorophore per cell was calculated in the spotted as well as the colocalized images using procedures in MetaMorph. The percentage colocalization was then calculated from the ratio of the net intensity within a cell in the two images. Percent colocalization is represented as a mean and SE obtained from two to three independent experiments with a minimum of 30 cells per experiment. All images were processed for output purposes using Adobe Photoshop software (Adobe).

For comparison of means between two groups, Student t test was performed. For multigroup comparisons, ANOVA test with a Bonferroni’s multiple comparison correction was used. All p values <0.05 were considered significant.

To examine the role of ligand density in CD8 T cell responses, we used naive CD44loCD62Lhi peripheral CD8 T cells from OT1 TCR-transgenic mice (38). Cells of an adherent epithelial cell line of H-2b MHC haplotype, PS-SW, which express modest levels of the costimulatory molecule CD80, were used as APCs. APCs were plated and prepulsed with titrating concentrations of SIINFEKL, the cognate peptide recognized by the OT1 TCR, prior to T cell–APC coculture. Titrating concentrations of SIINFEKL were used and generated graded concentrations of SIINFEKL–H-2Kb complexes as recognized by the mAb 25D1.16 (Supplemental Fig. 1A) (39). It is known that even on a transient exposure, naive CD8 T cells are programmed to undergo several rounds of division and differentiation without the continuous presence of ligand (17, 40, 41). We therefore cocultured T cells and APCs for 3 h before separating the adherent APCs from the T cells. T cells were then cultured without ligand for varying periods of time before assay. As expected, these cells showed early induction of activation marker molecules such as CD69 and CD25, and underwent multiple rounds of proliferation by 60 h (Supplemental Fig. 1B–D), leading to effector differentiation as measured by the ability to make IFN-γ upon restimulation (Supplemental Fig. 1E).

Using this system, with increasing ligand concentrations, the frequency of CD69+ responding OT1 cells was found to increase (Fig. 1A, 1B). However, at all ligand concentrations tested, the levels of CD69 expression on responding OT1 cells remained the same (Fig. 1C). Thus, as reported earlier (42, 43), the early activation response of individual naive CD8 T cells was binary. Increments in ligand density only increased the proportions of responding cells.

FIGURE 1.

Ligand density and metabolic parameters determine the size of the responding naive CD8 T cell population, but not CD69 levels on responding cells. (AC) OT1 cells were stimulated for 3 h with APCs pulsed with varying concentrations of SIINFEKL peptide (10,000 nM [continuous line], 100 nM [dark broken line], 1 nM [light broken line]), and then removed from APC monolayers and stained for CD69. Frequencies of CD69 expressing cells (B) and relative intensities of CD69 expression on CD69+ cells with reference to the intensities on CD69 cells (C) were calculated (mean ± SE, n = 4). (D) Titrating numbers of OT1 cells per well were stimulated for 3 h with APCs pulsed with various concentrations of SIINFEKL as shown, and frequencies of CD69+ cells were estimated (mean ± SE, n = 3). (EH) CD62LhiCD44lo CD8 OT1 T cells from OT1 [or OT1-GFP; for (F)] mice as necessary were further sort purified into the brightest 10% and the dullest 10% subsets for cell size; FSC, (E); GFP, (F); mitochondrial mass (as measured by Mitotracker Green staining intensity), (G); or glucose uptake (as measured by 2-NBDG intensity), (H). Purified cells were stimulated with APCs pulsed with varying concentrations of SIINFEKL peptide as indicated for 3 h, followed by staining for CD69. Frequencies of CD69 expressing cells are shown (mean ± SE, n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 1.

Ligand density and metabolic parameters determine the size of the responding naive CD8 T cell population, but not CD69 levels on responding cells. (AC) OT1 cells were stimulated for 3 h with APCs pulsed with varying concentrations of SIINFEKL peptide (10,000 nM [continuous line], 100 nM [dark broken line], 1 nM [light broken line]), and then removed from APC monolayers and stained for CD69. Frequencies of CD69 expressing cells (B) and relative intensities of CD69 expression on CD69+ cells with reference to the intensities on CD69 cells (C) were calculated (mean ± SE, n = 4). (D) Titrating numbers of OT1 cells per well were stimulated for 3 h with APCs pulsed with various concentrations of SIINFEKL as shown, and frequencies of CD69+ cells were estimated (mean ± SE, n = 3). (EH) CD62LhiCD44lo CD8 OT1 T cells from OT1 [or OT1-GFP; for (F)] mice as necessary were further sort purified into the brightest 10% and the dullest 10% subsets for cell size; FSC, (E); GFP, (F); mitochondrial mass (as measured by Mitotracker Green staining intensity), (G); or glucose uptake (as measured by 2-NBDG intensity), (H). Purified cells were stimulated with APCs pulsed with varying concentrations of SIINFEKL peptide as indicated for 3 h, followed by staining for CD69. Frequencies of CD69 expressing cells are shown (mean ± SE, n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal

We next tested the formal possibility that, at low ligand densities, access of monoclonal OT1 T cells to ligand was physically limited by cellular competition for ligand. To test this, we varied the input T cell numbers for each ligand concentration. However, even 10-fold variation in input OT1 cell numbers did not alter the frequency of T cells responding at a given ligand concentration (Fig. 1D). These data indicated that T cell sensitivity to cognate ligand is cell intrinsic, and that stimulus thresholds for activation vary even within a monoclonal CD8 T cell population.

Variation in the ability to respond to TCR-mediated stimulation in a monoclonal CD8 T cell population would be expected to correlate with and depend on variation in other properties of the cell population. We therefore tested a number of parameters for such correlation. To do this, we chose parameters detectable by flow cytometry in live cells, sort purified bright and dull deciles of the OT1 CD8 T cell population for that parameter, and compared the ligand dose–response relationships of the two subpopulations. Variation in cell size, a relatively crude marker of metabolic activity, did not correlate with the ability to respond to TCR-mediated stimulation (Fig. 1E, Supplemental Fig. 1F). However, when we specifically identified OT1 cells making low versus high levels of a heterologous protein, enhanced GFP (eGFP), using cells from OT1 mice also carrying a transgene for eGFP-expression, eGFPlo cells were less receptive than eGFPhi cells (Fig. 1F, Supplemental Fig. 1G), indicating that variations in cellular rates of protein synthesis may be involved in the ability to respond to TCR-mediated stimulation. Cellular mitochondrial mass, as estimated by the dye Mitotracker Green, did not show any correlation with the ability to respond to TCR-mediated stimulation (Fig. 1G, Supplemental Fig. 1H). However, when OT1 T cells were cultured with a fluorescent glucose analog, 2-NBDG, and subsequently sort purified for low versus high glucose uptake, NBDGlo cells showed a distinctly lower ability to respond to TCR-mediated stimulation as compared with NBDGhi cells (Fig. 1H, Supplemental Fig. 1I), suggesting that cell population variation in glucose metabolism could be involved in regulating the variation in the ability to respond to TCR-mediated stimulation.

Although the levels of CD69 expression induced on individual responding T cells appeared to be insensitive to ligand density, it remained possible that more complex downstream outcomes of activation, such as proliferation, may be quantitatively correlated to ligand density. In fact, data using stimulation in vivo have been used to argue that increasing ligand density leads to increasing proliferation (3), although the likelihood of repeated stimulation of T cells is a caveat for such studies. To examine if the proliferation program in briefly stimulated OT1 T cells was sensitive to ligand density, we purified CFSE-labeled CD69+ OT1 T cells after 3 h cocultures as above with APCs loaded with graded concentrations of cognate peptide. As expected, although their frequencies were quite different, the sort-purified CD69+ OT1 cells had almost identical CD69 expression levels at all ligand densities (Fig. 2A). Further, over the ligand density range tested, all sort-purified CD69+ T cells proliferated (Fig. 2B) and to approximately the same extent. However, in cells triggered with the lowest loading concentration of cognate peptide (1 nM), the proliferative response was delayed (Fig. 2C, 2D). This was not due to differential cell death in different cell generations, because dead cell frequencies in each division cycle were similar at high and low ligand densities (Fig. 2E, 2F). These data suggested that, although the proliferative program is also relatively insensitive to ligand densities, there is an initial delay in entering the proliferation program in OT1 T cells triggered with very low ligand densities.

FIGURE 2.

Delayed proliferation in low ligand density-stimulated CD8 T cells. (A) CFSE-labeled OT1 cells were stimulated with APCs pulsed with 1000 nM (continuous lines) or 1 nM (broken lines) SIINFEKL peptide, and CD69-expressing CD8 cells were sort purified 3 h later. (BF) These purified CD69-expressing CD8 T cells were cultured in medium (with 10 U/ml IL-2) and CFSE dilution analyzed 60 h later, for the frequencies of CFSE-diluted cells (B and C) and for detailed cell generation analysis (D) (mean ± SE, n = 4). The frequencies of live cells in culture (E) and the frequencies of dead cells in each cell generation (F) are also shown. (G) CD69-expressing OT1 cells were sort purified as above and cultured for a further 4–6 h without IL-2 to allow CD25 expression and then stained with anti-CD25 for estimating CD25 expression levels as mean fluorescence intensity (MFI) as shown (mean ± SE; n = 3). *p < 0.05, **p < 0.01.

FIGURE 2.

Delayed proliferation in low ligand density-stimulated CD8 T cells. (A) CFSE-labeled OT1 cells were stimulated with APCs pulsed with 1000 nM (continuous lines) or 1 nM (broken lines) SIINFEKL peptide, and CD69-expressing CD8 cells were sort purified 3 h later. (BF) These purified CD69-expressing CD8 T cells were cultured in medium (with 10 U/ml IL-2) and CFSE dilution analyzed 60 h later, for the frequencies of CFSE-diluted cells (B and C) and for detailed cell generation analysis (D) (mean ± SE, n = 4). The frequencies of live cells in culture (E) and the frequencies of dead cells in each cell generation (F) are also shown. (G) CD69-expressing OT1 cells were sort purified as above and cultured for a further 4–6 h without IL-2 to allow CD25 expression and then stained with anti-CD25 for estimating CD25 expression levels as mean fluorescence intensity (MFI) as shown (mean ± SE; n = 3). *p < 0.05, **p < 0.01.

Close modal

Because these cells were cultured in the presence of IL-2 after removal from APCs, we tested if different levels of CD25 were induced in OT1 cells triggered with low versus high ligand density. For this, CD69-expressing OT1 cells were sort purified as above and cultured for a further 4–6 h without IL-2 to allow CD25 expression and then stained with anti-CD25 for estimating CD25 expression levels. Although there was a tendency for low ligand density–triggered OT1 cells to express modestly lower levels of CD25, the difference did not reach robust statistical significance (Fig. 2G). The apparent delay in proliferation in low ligand density activated OT1 cells was thus unlikely to be due to induction of different levels of CD25.

We further investigated the delay in entering proliferation in low ligand density–triggered OT1 cells. At an early time point of 24/36 h postactivation, low ligand density–triggered OT1 cells were yet to show cell division, whereas many cells in the high ligand density–triggered group had already divided (Fig. 3A). We also tested if, consistent with such a delay, the frequencies of cells initiating DNA replication at an early time point postactivation were lower for low ligand density–triggered OT1 cells than for high ligand density–triggered ones. When OT1 cells in postactivation culture for 24 h were pulsed with EdU for 2 h, low ligand density–triggered OT1 cells indeed showed fewer cells incorporating EdU than high ligand density–triggered cells did (Fig. 3B, Supplemental Fig. 2A). Because the entry of T cells into cycle has been shown to be regulated by the cell cycle repressor p27kip1 (44), we tested for its loss in activated OT1 cells and found that, at 24 h postactivation, high ligand density–triggered OT1 cells showed greater loss of p27kip1 than low ligand density–triggered cells did (Fig. 3C, Supplemental Fig. 2A). Additionally, we tested the kinetics of induction of a crucial p27kip1 antagonist, c-myc, and found that induction of c-myc expression is more rapid in OT1 cells triggered with high ligand density than in those triggered by low ligand density (Fig. 3D, Supplemental Fig. 2).

FIGURE 3.

Correlates of delayed initiation of proliferation in low ligand density–stimulated CD8 T cells. OT1 cells were stimulated with APCs pulsed with 1000 nM (continuous lines) or 1 nM (broken lines) SIINFEKL peptide; CD69-expressing CD8 cells were sort purified 3 h later, and cultured in medium (with 10 U/ml IL-2) for varying periods of time. (A) CFSE dilution was assayed 24, 36, or 48 h later; the gray line at 24 h represents unstimulated CFSE-labeled cells for comparison. EdU incorporation (B), cellular levels of p27kip1 (C), or c-myc (D) were estimated 24 h later. 2-NBDG uptake estimation (E), pS6 level estimation (F), HPG incorporation assays (G), and cell size analysis (H) were done 12 h later (mean ± SE, n = 3–4). Cells were also incubated for longer periods instead, and 2-NBDG uptake estimation (I), pS6 level estimation (J), and HPG incorporation assays (K) were done 24 h later, whereas levels of c-myc (L) were estimated 36 h later (mean ± SE, n = 3–4). *p < 0.05.

FIGURE 3.

Correlates of delayed initiation of proliferation in low ligand density–stimulated CD8 T cells. OT1 cells were stimulated with APCs pulsed with 1000 nM (continuous lines) or 1 nM (broken lines) SIINFEKL peptide; CD69-expressing CD8 cells were sort purified 3 h later, and cultured in medium (with 10 U/ml IL-2) for varying periods of time. (A) CFSE dilution was assayed 24, 36, or 48 h later; the gray line at 24 h represents unstimulated CFSE-labeled cells for comparison. EdU incorporation (B), cellular levels of p27kip1 (C), or c-myc (D) were estimated 24 h later. 2-NBDG uptake estimation (E), pS6 level estimation (F), HPG incorporation assays (G), and cell size analysis (H) were done 12 h later (mean ± SE, n = 3–4). Cells were also incubated for longer periods instead, and 2-NBDG uptake estimation (I), pS6 level estimation (J), and HPG incorporation assays (K) were done 24 h later, whereas levels of c-myc (L) were estimated 36 h later (mean ± SE, n = 3–4). *p < 0.05.

Close modal

Upon activation, as CD8 T cells prepare for the energy-consuming process of cell division, they initiate increased protein synthesis (45) and begin to take up more extracellular glucose (46). We therefore tested these parameters, and observed that low ligand density–triggered OT1 cells showed delays in induction of increased glucose uptake as measured by labeled glucose uptake, in initiation of protein synthesis as measured by induction of phosphorylation of the ribosomal protein S6, in induction of increased rates of global protein synthesis as measured by HPG incorporation, and in the increase of cell size during the first 12 h postactivation (Fig. 3E–H, Supplemental Fig. 2A). Thus, naive CD8 T cells triggered by low ligand density initiate cellular responses more slowly than do cells triggered with high ligand density. However, when these groups of cells were incubated for longer periods, these parameters become comparable between the two groups (Fig. 3I–L), indicating that all such cells ultimately enter the cell cycle, proliferate, and differentiate, regardless of triggering ligand densities.

Naive CD8 T cells showed evidence of equivalent early activation by expressing comparable levels of CD69 at all triggering ligand densities, yet showed delayed entry into cell cycle if triggered by low ligand densities. Therefore, we tested the possibility that successfully activated (CD69-expressing) cells may make a subsequent decision based on ligand density. Because triggered TCRs are known to be rapidly internalized, in a process dependent on the level of TCR occupancy (10), we began by estimating cell surface TCR levels on triggered naive OT1 CD8 T cells. After a 3 h exposure to varying ligand densities, varying proportions of OT1 cells expressed CD69 as expected (Fig. 4A). At no ligand density was any loss of cell surface TCRs detected in cells not expressing CD69 (Fig. 4A), indicating that any decision of removal of cell surface TCRs occurs in cells that have made the binary decision to respond to the extent of CD69 induction. Notably, although CD69 levels on responding OT1 cells remained similar regardless of triggering ligand density, cell surface TCR levels varied depending on ligand density, and responding CD69-expressing cells showed no loss of cell surface TCRs at low density of ligand (Fig. 4A, 4B), suggesting that ligand density regulates an early postactivation decision of removal of cell surface TCRs, and that as ligand density increases, increasing proportions of the responding naive CD8 T cell population make the decision to remove cell surface TCRs.

FIGURE 4.

Ligand density regulates TCR internalization and time of cell cycle entry in activated CD8 T cells. (A and B) OT1 cells were stimulated with APCs pulsed with varying concentrations of SIINFEKL peptide as indicated, or left unstimulated (control). Cells were stained and analyzed for CD69 and TCR (linear scale) 3 h later (A). Cells were examined for CD69 expression and TCR levels and the frequencies of CD69-expressing cells showing low levels of TCR were calculated (B) (mean ± SE, n = 3–4). (CM) OT1 cells were stimulated with APCs pulsed with 1000 nM or 1 nM SIINFEKL peptide for 3 h, and CD69-expressing cells were further sort purified as TCRlo (continuous lines) and TCRhi (broken lines) subsets, and then cultured in medium (with 10 U/ml IL-2) for varying periods of time before analysis. CFSE dilution was analyzed after 72 h (C) (gray histogram represents unstimulated CFSE-labeled cells for comparison), and frequencies of cells in each cell generation were calculated (D). EdU incorporation was assayed after 24 h (E). Live cell frequencies were calculated after 72 h (F). Cellular levels of p27kip1 were estimated at 24 h by Western blotting (G) (each cell lyste was run at two concentrations as indicated), or by flow cytometry (H). Levels of c-myc (I) were estimated after 24 h. Cellular levels of pS6 (J), of HPG incorporation (K), cell size (L), and 2-NBDG uptake (M) were estimated after 12 h (mean ± SE, n = 3–4). *p < 0.05, **p < 0.01.

FIGURE 4.

Ligand density regulates TCR internalization and time of cell cycle entry in activated CD8 T cells. (A and B) OT1 cells were stimulated with APCs pulsed with varying concentrations of SIINFEKL peptide as indicated, or left unstimulated (control). Cells were stained and analyzed for CD69 and TCR (linear scale) 3 h later (A). Cells were examined for CD69 expression and TCR levels and the frequencies of CD69-expressing cells showing low levels of TCR were calculated (B) (mean ± SE, n = 3–4). (CM) OT1 cells were stimulated with APCs pulsed with 1000 nM or 1 nM SIINFEKL peptide for 3 h, and CD69-expressing cells were further sort purified as TCRlo (continuous lines) and TCRhi (broken lines) subsets, and then cultured in medium (with 10 U/ml IL-2) for varying periods of time before analysis. CFSE dilution was analyzed after 72 h (C) (gray histogram represents unstimulated CFSE-labeled cells for comparison), and frequencies of cells in each cell generation were calculated (D). EdU incorporation was assayed after 24 h (E). Live cell frequencies were calculated after 72 h (F). Cellular levels of p27kip1 were estimated at 24 h by Western blotting (G) (each cell lyste was run at two concentrations as indicated), or by flow cytometry (H). Levels of c-myc (I) were estimated after 24 h. Cellular levels of pS6 (J), of HPG incorporation (K), cell size (L), and 2-NBDG uptake (M) were estimated after 12 h (mean ± SE, n = 3–4). *p < 0.05, **p < 0.01.

Close modal

The data so far indicated that high ligand density mediated both cell surface TCR removal and rapid cell cycle entry in activated naive CD8 T cells. We therefore tested if the rapid cell cycle entry of OT1 cells by high ligand density could be entirely explained by the decision to remove cell surface TCRs. For this, OT1 cells were exposed to varying ligand levels for 3 h, and the CD69-expressing activated cells in each group were then sort purified into TCRlo and TCRhi subpopulations of cells which either had or had not removed cell surface TCRs respectively (Supplemental Fig. 3A). These sort-purified cells were further cultured for 60 h and the extent of proliferation estimated. Irrespective of the priming ligand density, TCRlo cells consistently showed faster proliferation than TCRhi cells (Fig. 4C, 4D), indicating that ligand density affected the rapidity of cell cycle entry via the decision to remove cell surface TCRs. Consistent with this, the frequencies of OT1 cells initiating DNA replication were higher in TCRlo than in TCRhi cells at early time points, again regardless of priming ligand densities (Fig. 4E, Supplemental Fig. 2C). This difference was not a result of differential cell death in TCRhi versus TCRlo cells, because cell death was equivalent (Fig. 4F).

Further, as observed earlier for high ligand density–triggered OT1 cells, TCRlo OT1 cells showed greater loss of p27kip1 and higher levels of c-myc than in TCRhi cells from the same population (Fig. 4G–I, Supplemental Fig. 2C). Similarly, such TCRlo OT1 cells showed rapid induction of initiation of protein synthesis as measured by induction of phosphorylation of the ribosomal protein S6, by labeled amino acid incorporation and by increase of cell size during the first 12 h postactivation compared with TCRhi cells (Fig. 4J–L), although the increase in glucose uptake was similar between the two subsets (Fig. 4M).

We next tested if these effects of low ligand density could be mimicked by a low affinity ligand. We used a mutant of the nominal SIINFEKL (N4) peptide, SIIQFEKL (Q4), which is known to have equivalent MHCI-binding affinity but low TCR affinity (9). In keeping with its low TCR affinity, Q4-loaded APCs induced lower CD69+ responding frequencies in OT1 cells at 3 h of exposure than equivalent N4-loaded APCs did (Fig. 5A). The levels of CD69 induced in responding cells were not, however, different between N4- versus Q4-triggered OT1 cells (Fig. 5B). Further, Q4-triggered OT1 cells responding by CD69 expression did not show cell surface TCR removal at any peptide concentration (Fig. 5C, 5D, Supplemental Fig. 3A). In fact, even 12 h incubation did not show any cell surface TCR loss in either low density N4-triggered or in Q4-triggered OT1 cells (Supplemental Fig. 3B).

FIGURE 5.

Low affinity cognate interactions fail to trigger cell surface TCR removal. OT1 cells were stimulated with APCs pulsed with varying concentrations of either SIINFEKL (N4) or SIIQFEKL (Q4) peptide as indicated. Cells were stained and analyzed for CD69 and TCR (linear scale) 3 h later (AC). Cells were examined for CD69 expression and TCR levels, and the frequencies of CD69-expressing cells calculated (A) (gray histogram represents unstimulated cells for comparison), and the intensities of CD69 on them estimated (B). The frequencies of CD69-expressing cells showing low levels of TCR were also calculated (D). Mean ± SE, n = 3–4. **p < 0.01, ***p < 0.001.

FIGURE 5.

Low affinity cognate interactions fail to trigger cell surface TCR removal. OT1 cells were stimulated with APCs pulsed with varying concentrations of either SIINFEKL (N4) or SIIQFEKL (Q4) peptide as indicated. Cells were stained and analyzed for CD69 and TCR (linear scale) 3 h later (AC). Cells were examined for CD69 expression and TCR levels, and the frequencies of CD69-expressing cells calculated (A) (gray histogram represents unstimulated cells for comparison), and the intensities of CD69 on them estimated (B). The frequencies of CD69-expressing cells showing low levels of TCR were also calculated (D). Mean ± SE, n = 3–4. **p < 0.01, ***p < 0.001.

Close modal

We then compared the cell proliferation kinetics in Q4-triggered OT1 cells with those in N4-triggered cells. CFSE-labeled CD69+ CD8 OT1 T cells were purified after 3 h exposure to N4- or Q4-pulsed APCs (Fig. 6A) and were further cultured as earlier. Q4-triggered OT1 cells showed a lag in proliferation in comparison with N4-triggered cells (Fig. 6B, 6C), similar to that seen above in cells triggered with low ligand density. This was not a consequence of differential cell survival, because cell death frequencies did not differ between N4-triggered and Q4-triggered cells undergoing proliferation (Fig. 6E). Further, the frequencies of OT1 cells initiating DNA replication at an early time point postactivation were lower for Q4-triggered than for N4-triggered OT1 cells when assayed using EdU pulsing at 24 h (Fig. 6D, Supplemental Fig. 2B). Thus, either low density of ligand or low affinity for ligand leads to lack of cell surface TCR removal and slow cell cycle entry in responding naive CD8 T cells.

FIGURE 6.

Low affinity cognate interactions fail to trigger rapid cell cycle entry. CTV-labeled OT1 cells were stimulated with APCs pulsed with 100 nM N4 peptide (continuous lines) or 100 nM Q4 peptide (broken lines) for 3 h, and CD69-expressing cells were sort purified (A) (gray histogram represents unstimulated cells for comparison), and cultured in medium (with 10 U/ml IL-2) for varying periods of time before analysis. CTV dilution was analyzed after 48 h (B) (gray histogram represents unstimulated CTV-labeled cells for comparison), and frequencies of cells in each cell generation were calculated (C). EdU incorporation was assayed after 24 h (D). Live cell frequencies were calculated after 48 h (E). Cellular levels of p27kip1 (F) and c-myc (G) were estimated after 24 h. Uptake of 2-NBDG (H), levels of pS6 (I), HPG incorporation (J), and cell size (K) were estimated after 12 h (mean ± SE, n = 3–4). Cells were also incubated for longer periods instead, and 2-NBDG uptake estimation (L), pS6 level estimation (M), and HPG incorporation assays (N) were done 24 h later, whereas levels of c-myc (O) were estimated 36 h later (mean ± SE, n = 3–4). *p < 0.05.

FIGURE 6.

Low affinity cognate interactions fail to trigger rapid cell cycle entry. CTV-labeled OT1 cells were stimulated with APCs pulsed with 100 nM N4 peptide (continuous lines) or 100 nM Q4 peptide (broken lines) for 3 h, and CD69-expressing cells were sort purified (A) (gray histogram represents unstimulated cells for comparison), and cultured in medium (with 10 U/ml IL-2) for varying periods of time before analysis. CTV dilution was analyzed after 48 h (B) (gray histogram represents unstimulated CTV-labeled cells for comparison), and frequencies of cells in each cell generation were calculated (C). EdU incorporation was assayed after 24 h (D). Live cell frequencies were calculated after 48 h (E). Cellular levels of p27kip1 (F) and c-myc (G) were estimated after 24 h. Uptake of 2-NBDG (H), levels of pS6 (I), HPG incorporation (J), and cell size (K) were estimated after 12 h (mean ± SE, n = 3–4). Cells were also incubated for longer periods instead, and 2-NBDG uptake estimation (L), pS6 level estimation (M), and HPG incorporation assays (N) were done 24 h later, whereas levels of c-myc (O) were estimated 36 h later (mean ± SE, n = 3–4). *p < 0.05.

Close modal

Expectedly, Q4-triggered OT1 cells showed slower loss of p27kip1 (Fig. 6F) and lower levels of c-myc than N4-triggered OT1 cells (Fig. 6G). They also showed a slower increase in glucose uptake, slower induction of phosphorylation of the ribosomal protein S6 and of labeled amino acid incorporation, and slower increase of cell size during the first 12 h postactivation compared with N4-triggered cells (Fig. 6H–K). However, once again, when these N4- versus Q4-activated cells were incubated for longer periods, these parameters become comparable between the two groups (Fig. 6L–O), indicating that all such cells ultimately enter the cell cycle, proliferate, and differentiate, regardless of triggering ligand affinities.

In order to examine the possibility that these findings depend on nonconventional epithelial cell APCs, we tested peptide-pulsed BMDCs to activate OT1 CD8 T cells. Titrating concentrations of SIINFEKL generated graded concentrations of SIINFEKL–H-2Kb complexes (Fig. 7A). When OT1 cells were cocultured with peptide-pulsed BMDCs for 3 h, the frequency of CD69-expressing OT1 cells increased with increasing peptide concentration (Fig. 7B), but CD69 levels on responding OT1 cells were comparable at all peptide concentrations (Fig. 7C). Variation in BMDC:OT1 cell ratios did not alter the frequencies of OT1 cells responding at a given ligand concentration (Fig. 7D). At low ligand densities, responding CD69-expressing OT1 cells showed no TCR internalization (Fig. 7E, 7F).

FIGURE 7.

Conventional BMDCs as APCs mediate similar responses from OT1 T cells. (A) BMDCs were pulsed for 6 h with SIINFEKL peptide (1000 nM [continuous line], 1 nM [broken line]), washed, and stained with the SIINFEKL–H-2Kb complex-specific mAb 25D1.16. Gray histogram indicates unpulsed control. (B and C) OT1 cells were stimulated for 3 h with BMDCs pulsed with varying concentrations of SIINFEKL peptide (1000 nM [continuous lines], 1 nM [broken lines]), and then removed from APC monolayers and stained for CD69 and TCR. Frequencies of CD69-expressing cells (B) and relative intensities of CD69 expression on gated CD69-expressing cells (C) were calculated. Gray histograms represent unstimulated cells for comparison (mean ± SE, n = 3). (D) Titrating ratios as shown of OT1 cells and BMDCs prepulsed with various concentrations of SIINFEKL were cocultured for 3 h, and frequencies of CD69-expressing cells were estimated (mean ± SE, n = 3). (E and F) OT1 cells were stimulated for 3 h with BMDCs pulsed with 1000 nM or 1 nM SIINFEKL peptide, and then examined for CD69 expression and TCR levels (E), and the frequencies of CD69-expressing cells showing low levels of TCR were calculated (F) (mean ± SE, n = 3). (GJ) OT1 cells were stimulated for 3 h with BMDCs pulsed with 100 nM SIINFEKL (N4) or SIIQFEKL (Q4) peptide. Cells were examined for CD69 expression and the frequencies of CD69-expressing cells calculated (G) and the intensities of CD69 on the gated CD69-expressing cells were estimated (H). Gray histograms represent unstimulated cells for comparison. Cells were also examined for CD69 expression and TCR levels (I), and the frequencies of CD69-expressing cells showing low levels of TCR were calculated (J) (mean ± SE, n = 3). (K) CTV-labeled OT1 cells were stimulated for 3 h with APCs pulsed with 1000 nM or 1 nM N4 peptide, CD69-expressing cells were sort purified and cultured in medium (with 10 U/ml IL-2), and CTV dilution was analyzed after 48 h. *p < 0.05, **p < 0.01.

FIGURE 7.

Conventional BMDCs as APCs mediate similar responses from OT1 T cells. (A) BMDCs were pulsed for 6 h with SIINFEKL peptide (1000 nM [continuous line], 1 nM [broken line]), washed, and stained with the SIINFEKL–H-2Kb complex-specific mAb 25D1.16. Gray histogram indicates unpulsed control. (B and C) OT1 cells were stimulated for 3 h with BMDCs pulsed with varying concentrations of SIINFEKL peptide (1000 nM [continuous lines], 1 nM [broken lines]), and then removed from APC monolayers and stained for CD69 and TCR. Frequencies of CD69-expressing cells (B) and relative intensities of CD69 expression on gated CD69-expressing cells (C) were calculated. Gray histograms represent unstimulated cells for comparison (mean ± SE, n = 3). (D) Titrating ratios as shown of OT1 cells and BMDCs prepulsed with various concentrations of SIINFEKL were cocultured for 3 h, and frequencies of CD69-expressing cells were estimated (mean ± SE, n = 3). (E and F) OT1 cells were stimulated for 3 h with BMDCs pulsed with 1000 nM or 1 nM SIINFEKL peptide, and then examined for CD69 expression and TCR levels (E), and the frequencies of CD69-expressing cells showing low levels of TCR were calculated (F) (mean ± SE, n = 3). (GJ) OT1 cells were stimulated for 3 h with BMDCs pulsed with 100 nM SIINFEKL (N4) or SIIQFEKL (Q4) peptide. Cells were examined for CD69 expression and the frequencies of CD69-expressing cells calculated (G) and the intensities of CD69 on the gated CD69-expressing cells were estimated (H). Gray histograms represent unstimulated cells for comparison. Cells were also examined for CD69 expression and TCR levels (I), and the frequencies of CD69-expressing cells showing low levels of TCR were calculated (J) (mean ± SE, n = 3). (K) CTV-labeled OT1 cells were stimulated for 3 h with APCs pulsed with 1000 nM or 1 nM N4 peptide, CD69-expressing cells were sort purified and cultured in medium (with 10 U/ml IL-2), and CTV dilution was analyzed after 48 h. *p < 0.05, **p < 0.01.

Close modal

Further, Q4-pulsed BMDCs induced lower frequencies of responding OT1 cells than N4-pulsed BMDCs did at 3 h of exposure (Fig. 7G). CD69 levels on these responding OT1 cells were not, however, different between the N4- versus Q4-triggered cell populations (Fig. 7H). Q4-triggered OT1 cells did not show TCR internalization even at high ligand density (Fig. 7I, 7J). Finally, OT1 cells activated by low ligand density proliferated more slowly compared with cells responding to high ligand density (Fig. 7K). Thus, the density- and affinity-dependent modulation of OT1 CD8 T cell responses was independent of APC type.

Because OT1 T cells triggered with either high ligand density or with a high affinity ligand showed efficient cell surface TCR removal and rapid cell cycle entry, we tested the possibility that internalized TCRs may continue to signal, resulting in faster entry into cell cycle. If so, this signaling may continue at the same time as the internalized TCRs traverse through various compartments of the endolysosomal pathway before degradation in the lysosomes. We reasoned that, if such were the case, even after their removal from APC contact after a 3 h exposure, membrane-proximal signaling would still be operative in N4 ligand–triggered CD69+ OT1 cells that were TCRlo but not in those that were TCRhi, and not in cells triggered with the Q4 ligand. We tested this by treating the activated and purified cells during the first 4 h of postexposure culture with a PI3K inhibitor to block an essential early step downstream of TCR- and CD28-mediated signaling. Such transient treatment with the PI3K inhibitor postactivation led to delayed proliferation profiles in OT1 cells that were triggered with N4 peptide and were TCRlo, but did not affect either N4 ligand–triggered TCRhi cells or Q4 ligand–triggered cells (Fig. 8A, 8B). Thus, TCR-mediated signaling cascades persist after APC removal in responding CD8 T cells with internalized TCRs.

FIGURE 8.

Prolonged signaling downstream of putatively internalized TCRs leads to faster cell cycle entry in responding T cells. (A) CTV-labeled OT1 cells were stimulated with APCs pulsed with 1000 nM N4 peptide or 1000 nM Q4 peptide for 3 h; CD69-expressing cells were sort purified and cultured in medium (with 10 U/ml IL-2) for 4 h in the presence (broken line) or absence (continuous line) of the PI3K inhibitor (LY294002, 5 μM), then washed and further cultured until 48 h, and analyzed for CTV dilution. Three rows represent data from three independent experiments. (B) CTV-labeled OT1 cells were stimulated with APCs pulsed with N4 peptide for 3 h; CD69-expressing TCRhi and TCRlo cells were sort purified and cultured in medium (with 10 U/ml IL-2) for 4 h in the presence (broken line) or absence (continuous line) of the PI3K inhibitor (LY294002, 5 μM), then washed and further cultured until 48 h, and analyzed for CTV dilution. Three rows represent data from three independent experiments. (C) Control or DN-Rab7–transfected B3Z cells were stimulated with APCs pulsed with N4 (1000 nM) or Q4 (1000 nM) peptides for 3 h, removed, and put in APC-free culture for 10 h, and induction of bGal in B3Z cells was assayed (mean ± SE, n = 3). **p < 0.01.

FIGURE 8.

Prolonged signaling downstream of putatively internalized TCRs leads to faster cell cycle entry in responding T cells. (A) CTV-labeled OT1 cells were stimulated with APCs pulsed with 1000 nM N4 peptide or 1000 nM Q4 peptide for 3 h; CD69-expressing cells were sort purified and cultured in medium (with 10 U/ml IL-2) for 4 h in the presence (broken line) or absence (continuous line) of the PI3K inhibitor (LY294002, 5 μM), then washed and further cultured until 48 h, and analyzed for CTV dilution. Three rows represent data from three independent experiments. (B) CTV-labeled OT1 cells were stimulated with APCs pulsed with N4 peptide for 3 h; CD69-expressing TCRhi and TCRlo cells were sort purified and cultured in medium (with 10 U/ml IL-2) for 4 h in the presence (broken line) or absence (continuous line) of the PI3K inhibitor (LY294002, 5 μM), then washed and further cultured until 48 h, and analyzed for CTV dilution. Three rows represent data from three independent experiments. (C) Control or DN-Rab7–transfected B3Z cells were stimulated with APCs pulsed with N4 (1000 nM) or Q4 (1000 nM) peptides for 3 h, removed, and put in APC-free culture for 10 h, and induction of bGal in B3Z cells was assayed (mean ± SE, n = 3). **p < 0.01.

Close modal

We also tested this possibility by over-expressing a DN form of Rab7 (DN-Rab7) protein in the T cell hybridoma B3Z, which expresses the same TCR that OT1 cells do. Rab7 is a protein involved in cargo trafficking from early to late endosomes. Over-expression of DN-Rab7 in B3Z, therefore, would dominantly inhibit the cargo delivery from early to late endosomes and subsequently delay the degradation of endocytosed TCRs in triggered B3Z cells. Indeed, DN-Rab7–expressing B3Z cells responded better upon limited time (3 h) stimulation with N4 ligand, but not if similarly stimulated by the low affinity Q4 ligand, as measured by expression of IL-2 promoter-driven bGal reporter protein (Fig. 8C). These data suggested that internalized TCRs continue to signal until they reach late endosomes/lysosomes.

In order to examine further the potential relationship between activation-induced TCR endocytosis and continued signaling, we used transient transfection of B3Z cells with DN or DA variant versions of a number of molecules involved in endocytosis, followed by transient activation of these B3Z cells on SIINFEKL-pulsed PS-SW cells, followed in turn by detection of the intracellular expression of the NFAT-dependent activation reporter bGal.

A 2 h exposure to peptide-pulsed APCs, followed by removal and culture, led to transient bGal expression in B3Z cells detectable starting at 10 h postactivation and becoming near undetectable by ∼25 h postactivation (Fig. 9A). The frequencies of bGal-expressing B3Z cells increased with increasing pulsing peptide concentrations (Fig. 9A), but the extent of bGal expression per cell in responding cells remained constant regardless of ligand density (Fig. 9A). These data were consistent with the data using OT1 CD8 T cells (Fig. 1), and indicated that ligand concentration determined the frequency of responding cells but that the responses of individual cells were binary.

FIGURE 9.

Modification of signaling kinetics in B3Z cells postactivation upon alteration of endolysosomal trafficking. (A) B3Z cells were stimulated for 2 h with PS-SW APCs prepulsed with SIINFEKL at 30 ng/ml (black lines), 300 ng/ml (gray continuous lines), or 3000 ng/ml (gray broken lines). B3Z cells were removed from APCs and cultured for varying periods as shown before flow cytometric detection of intracellular bGal. Gray-filled histograms represent unstimulated B3Z cells. Data are representative of >10 independent experiments. (B) B3Z cells were transiently transfected with constructs for expressing various endolysosomal trafficking modifiers as indicated with GFP tags. Either WT (open symbols) or DN (black-filled symbols) versions of Rab11, Eps15, Dynamin-2, Rab21, Rab22, Rab5, and Rab7 were used. A DA version (gray-filled symbols) of Rab5 was also tested. For AP180, either GFP alone (open symbols) or the AP180C version (black-filled symbols) was used. Transfected cells were stimulated as in (A) above with APCs pulsed with 300 ng/ml SIINFEKL (30 ng/ml for the Rab7 panel), removed from APCs, and cultured for varying periods of time as indicated before being stained for bGal. Frequencies of bGal-expressing GFP+ cells are shown. Data are representative of two to four independent experiments for each panel.

FIGURE 9.

Modification of signaling kinetics in B3Z cells postactivation upon alteration of endolysosomal trafficking. (A) B3Z cells were stimulated for 2 h with PS-SW APCs prepulsed with SIINFEKL at 30 ng/ml (black lines), 300 ng/ml (gray continuous lines), or 3000 ng/ml (gray broken lines). B3Z cells were removed from APCs and cultured for varying periods as shown before flow cytometric detection of intracellular bGal. Gray-filled histograms represent unstimulated B3Z cells. Data are representative of >10 independent experiments. (B) B3Z cells were transiently transfected with constructs for expressing various endolysosomal trafficking modifiers as indicated with GFP tags. Either WT (open symbols) or DN (black-filled symbols) versions of Rab11, Eps15, Dynamin-2, Rab21, Rab22, Rab5, and Rab7 were used. A DA version (gray-filled symbols) of Rab5 was also tested. For AP180, either GFP alone (open symbols) or the AP180C version (black-filled symbols) was used. Transfected cells were stimulated as in (A) above with APCs pulsed with 300 ng/ml SIINFEKL (30 ng/ml for the Rab7 panel), removed from APCs, and cultured for varying periods of time as indicated before being stained for bGal. Frequencies of bGal-expressing GFP+ cells are shown. Data are representative of two to four independent experiments for each panel.

Close modal

When B3Z cells were transiently transfected to express various DN, DA, or WT versions of trafficking-related molecules along with eGFP before being transiently stimulated with peptide-pulsed APCs, the time course of the B3Z response to stimulation was significantly altered in a number of instances. Thus, variant versions shown to inhibit internalization itself—namely AP180C, DN-Eps15, DN-dyn [as previously shown (47)], and DN-Rab5—all showed early enhancement of the responses of B3Z cells (Fig. 9B). Interestingly, variant versions that have been shown to inhibit postinternalization endosomal trafficking and lysosomal fusion—namely, DN-Rab5, DN-Rab7, DN-Rab21, and DN-Rab22—all showed somewhat later enhancement of the responses of B3Z cells (Fig. 9B). DN-Rab11, which was not expected to modify endolysosomal trafficking, did not alter B3Z responses (Fig. 9B).

We next confirmed the expected effects of some of these molecules on TCR internalization and/or delivery to LAMP-1–bearing vesicular degradatory compartments, using confocal microscopy. Transfected B3Z cells were stimulated with peptide-pulsed APCs for varying durations, then permeabilized and stained, and eGFP-expressing cells were imaged for TCR and LAMP-1 distribution (Supplemental Fig. 4), with data quantification (Fig. 10A, 10B). As expected, DN-Eps15 and DN-Rab5 caused delayed TCR endocytosis whereas DN-Rab7 did not (Fig. 10A). On the other hand, when the colocalization of endocytosed TCRs with LAMP-1 was quantified, it was evident that DN-Eps15 did not affect trafficking of internalized TCR, whereas DN-Rab5 and DN-Rab7 both delayed it (Fig. 10B).

FIGURE 10.

TCR trafficking and signaling postactivation in B3Z cells. B3Z cells were transiently transfected with constructs for expressing various endolysosomal trafficking modifiers as indicated with GFP tags. Either WT or DN versions of Eps15, Rab5, or Rab7 were used, along with a GFP-alone construct and an untransfected B3Z control (unT). Transfected cells were stimulated with PS-SW APCs prepulsed with SIINFEKL (300 ng/ml) for varying periods of time as shown before being fixed, permeabilized, stained, and imaged by confocal microscopy as described. TCR internalization (A), colocalization of internalized TCRs with LAMP-1 (B), and colocalization of internalized TCRs with phospho-ZAP70 (C) were quantified over time as shown. Mean ± SE, n = ∼30 cells per point.

FIGURE 10.

TCR trafficking and signaling postactivation in B3Z cells. B3Z cells were transiently transfected with constructs for expressing various endolysosomal trafficking modifiers as indicated with GFP tags. Either WT or DN versions of Eps15, Rab5, or Rab7 were used, along with a GFP-alone construct and an untransfected B3Z control (unT). Transfected cells were stimulated with PS-SW APCs prepulsed with SIINFEKL (300 ng/ml) for varying periods of time as shown before being fixed, permeabilized, stained, and imaged by confocal microscopy as described. TCR internalization (A), colocalization of internalized TCRs with LAMP-1 (B), and colocalization of internalized TCRs with phospho-ZAP70 (C) were quantified over time as shown. Mean ± SE, n = ∼30 cells per point.

Close modal

Finally, we confirmed enhanced and persistent signaling from engaged and internalized TCRs by stimulating transfected B3Z cells with peptide-pulsed APCs for various times, then permeabilizing and staining them for TCR and phospho-ZAP70, and imaging eGFP-expressing cells by confocal microscopy (Supplemental Fig. 4), and quantifying the TCR-phospho-ZAP70 colocalization. The total phospho-ZAP70 signal per cell was higher in B3Z cells expressing DN-Eps15, DN-Rab5, and DN-Rab7, it remained high even at 30 min postactivation in cells expressing DN-Rab5 or DN-Rab7, and colocalized with TCR-positive vesicles (Fig. 10C). Together, these data established that postactivation TCR internalization had major functional consequences for the T cells as a result of quantitative trafficking-dependent modulation of continued signaling from endocytosed TCRs.

T cells encounter cognate antigenic peptide targets on APCs within lymphoid tissues. T cell activation is commonly thought of as a trigger at a single point in time that leads to activation-related decisions in T cells, which, if successful, induce a program of proliferation and effector differentiation. Yet, it is evident that, in an environment where cognate ligand is present, an individual T cell is likely to have multiple cognate encounters separated in time. This can occur at two distinct levels. In one, T cells move from APC to APC immediately upon first encounter with cognate ligand (18), and are thus likely to have a series of discrete cognate encounters within minutes to a couple of hours. Secondly, cognate T cell clones that have begun to respond by proliferation and differentiation will frequently continue to be located in the cognate ligand environment and will therefore continue to have cognate encounters at various times during and after the proliferation–differentiation program. Although T cell response magnitudes increase with ligand dose and affinity (39), the question of how responding T cells detect ligand density and/or affinity is complicated by the likelihood of serial cognate encounters.

We have therefore taken advantage of the fact that naive CD8 T cells can initiate an autonomous program of proliferation and differentiation upon brief encounter with an antigenic stimulus, and go through such a program in the absence of Ags (17, 40, 41), to restrict cognate encounters to a very short period to ask how specific CD8 T cells respond to quantitatively varying ligand density or ligand affinity. Our data show that ligand density- or affinity-mediated increase in T cell response magnitude depends on variation in activation thresholds even among a monoclonal CD8 T cell population, and that this activation threshold may be dependent on variation in basal metabolism. Further, our data show that high ligand density or affinity leads to enhanced TCR internalization in responding CD8 T cells, and this internalization is associated, via sustained signaling, with more rapid entry into the proliferation program. In fact, the strict dose dependence of all tested features of the activation-proliferation program of naive CD8 T cells here suggests that they are responsive to determination by the law of mass action governing encounters between TCRs and ligands, as has been indicated for effector CD8 T cells (48).

Conventional and nonconventional APCs can differ widely in cell size, plasma membrane cholesterol content, levels and distribution of adhesion and costimulatory molecules, MHCI expression levels, and self-peptide repertoires, as well as pMHCI clustering patterns, all issues that can plausibly affect ligand detection by T cells (4952). However, findings observed using PS-SW epithelial cells as APCs were reproducible using BMDCs as APCs, supporting the interpretation of a role for ligand density or affinity in mediating the quantitative variations we have observed. Although it was possible that OT1 cells exposed briefly to peptide-pulsed APCs themselves could then carry over ligand for continued stimulation in APC-free cultures, we tested but could not detect any evidence for this possibility (Supplemental Fig. 3C, 3D).

T cells undergo substantial metabolic changes upon cognate encounter (53). As they prepare to initiate cell cycle, aerobic glycolysis becomes the prominent feature of glucose catabolism (54) and T cells deficient in glucose transporter Glut1 fail to elicit effector functions (46). Protein synthesis is upregulated to increase cell mass preparatory to cell division (45). It is therefore plausible to imagine that variation in baseline cellular metabolic status may provide the basis for the differences in activation threshold of individual T cells. Our data show that CD8 T cells in the peripheries of a cell population unimodally distributed for efficiency of either glucose uptake or marker protein turnover show differences in their activation thresholds, so that high efficiency cells are more responsive to ligand. Thus, the metabolic competence of responding T cells may be a critical factor determining their response to activation, and variation in cellular metabolism may be a major regulator of ligand-T cell dose-response relationships.

The mechanisms causing such variability in homogenous monoclonal naive T cell populations are likely to be a mixture of the stochastic and the environmentally induced. Nongenetic cell-to-cell variability can arise due to intrinsic heterogeneity in the level of gene expression of molecules that are involved in basal metabolism and TCR signaling pathways. Because expression of each gene is controlled by the concentrations and locations of multiple transcription factors, polymerases, and other regulatory proteins, fluctuations in the amount or activity of these molecules can cause corresponding fluctuations in the output of the gene (55). This biochemical noise in gene expression has been implicated to result in phenotypic noise in clonal cellular populations; specifically, intraclonal differences in the expression of CD8 and the tyrosine phosphatase SHP-1 have been shown to generate dispersion in responsiveness among individual CD8 T cells (56). This variability is likely to be a major cell-intrinsic mechanism regulating ligand-T cell dose–response relationships.

It is also possible that some cell-extrinsic microenvironmental mechanisms may be involved as well. We have recently shown that subsets with differing phenotypes and effector functions can arise in unimodally distributed naive CD4 T cell populations as a function of time spent in the peripheral lymphoid organs (57), and variations in the duration of tonic TCR interactions with self-pMHC could contribute to inherent heterogeneity in Ag sensing of naive T cells (58).

The CD8 T cells that do respond to various ligand densities appear to exhibit binary early activation. Thus, the frequency of CD69-expressing cells changes with ligand density or affinity, but the levels of CD69 expression in individual responding cells remain constant. This is consistent with data showing that the MAPK-ERK signaling module downstream of the TCR follows such a binary pattern in which, with increasing stimulus concentrations, an increasingly large fraction of the T cell population acquires active phosphorylated ERK molecules, but the amounts of phosphorylated ERK per cell remain constant (42, 43, 59, 60).

In fact, the proliferation–differentiation program of those CD8 T cells that do turn on CD69 expression at any ligand density or affinity appears unchanged by the density or affinity of the triggering ligand, in that they all proliferate and differentiate. However, the speed of entry into cell cycle after activation is related to the density or affinity of ligand exposure. Thus, although all CD69-expressing cells express similar levels of CD69 and proliferate and differentiate, the rate at which they undertake metabolic changes and enter cell cycle is faster in high ligand density/affinity–triggered cells, so that they are one generation ahead in proliferation. They show high levels of protein synthesis, of c-myc expression, and of DNA replication at earlier time points. Thus, ligand density and/or affinity determine the proportion of responding CD8 T cells and the speed with which they respond, but the actual core proliferation–differentiation program individual responding CD8 T cells execute is insensitive to ligand density or affinity. These data confirm and extend previous findings showing the existence of a signaling threshold for activation and, with a temporal component, for proliferation that is independent of signal magnitude (12).

How do responding T cells with equivalent CD69 expression modulate the rate of entry into the proliferation–differentiation program in response to ligand density or affinity? Our data show that, even when different ligand densities or affinities trigger responding CD8 T cells with the same levels of CD69 expression, these T cells show differences in the extent of cell surface TCR removal. T cells primed with higher ligand density or affinity lose more TCR from the surface. TCR removal from the cell surface during T cell activation has been shown to be a consequence of TCR internalization from the cognate immunological synapse, and is commonly thought of as a mechanism to terminate TCR-mediated signaling (6164). The biochemical factors regulating the efficiency of cognate ligand-induced TCR internalization are not well understood, although high TCR occupancy (10) undoubtedly plays a significant role, and our data indicate that this is mediated via high ligand density or affinity. Again, our data confirm and extend previous findings indicating that the affinities of TCR ligands determines the rate at which the cumulative threshold signal for T cell activation is reached (65); the present data show that differential TCR internalization and continued signaling is one determinant of this rate.

Interestingly, CD69-expressing responding T cells with internalized TCRs show rapid metabolic reprogramming and cell cycle entry, regardless of the ligand density or affinity at which they are triggered. Thus, ligand density or affinity determine the extent of TCR internalization in individual responding CD8 T cells, and TCR internalization in turn modulates the rate of entry of the T cell into the proliferation–differentiation program. The endolysosomal system, once associated only with degradation, is now recognized as a novel cellular platform orchestrating multiple signaling pathways so that internalized TCRs may continue to signal via kinases associated with internalized TCR complexes in endocytic vesicles (66), just as internalized B cell receptors do, or indeed other receptor species such as the epidermal growth factor receptor (67, 68). There are suggestions that internalized TCRs may take pMHC complexes from APCs with them into the T cell via trogocytosis (69), possibly mediating continued signaling.

Our data are consistent with a model of continued signaling from internalized TCRs, allowing for more prolonged signaling in TCRlo versus TCRhi cells resulting in accelerated entry into the proliferation–differentiation program. In support of this, we find that when CD69-expressing CD8 T cells are treated with a PI3K inhibitor after removal of the cognate ligand, their entry into cell cycle is slower only if they have internalized TCRs, but not if they have not done so. This indicates that TCR internalization is associated with continued signaling involving PI3K that is responsible for rapid initiation of the proliferation program. Thus, our data support a model of internalized TCRs continuing to signal, although it remains possible, of course, that TCR signals from the plasma membrane versus endosomes are qualitatively distinct, as has been suggested for TLR4 (70).

It is interesting to note that one major difference shown by our data between weak and strong TCR ligands is the speed at which responding naive CD8 T cells begin to proliferate. This is likely to have functional consequences during an infection in vivo. Early during infection, at low ligand densities, high affinity CD8 T cells would begin to respond and proliferate rapidly, as well as possibly beginning to remove ligands from APC surfaces, thereby occupying more and more of the responding pool of T cells, leading to interclonal affinity maturation-driven oligoclonality. However, very low levels of ligand and/or the absence of high affinity TCRs in the naive pool will lead to delayed proliferation and persistent recruitment and slow expansion of a clonally diverse responding population. The functional consequences of these distinctions for effector versus long-term memory functions of primed antiviral protective CD8 T cell populations will be interesting to examine further.

If internalized TCRs can continue to signal, an obvious cell biological point for signal termination would be at endolysosomal fusion. A prediction from such a possibility would be that inhibition of endolysosomal fusion and/or lysosomal degradation would lead to prolonged TCR signaling in T cells in which TCRs are internalized, but not in those with no TCR internalization. A DN version of the Rab7 gene (DN-Rab7) inhibits the critical Rab7 functions for endolysosomal fusion leading to delayed fusion (71). Our data using a DN-Rab7–expressing T cell hybridoma expressing the same TCR as OT1 T cells show that, consistent with this model, inhibition of Rab7 function enhances the response of the T cell hybridoma to high affinity ligand but not to low affinity ligand.

Further, our detailed experiments with the B3Z hybridoma confirm and extend the bulk of these findings and inferences, albeit with the major formal caveat concerning the uncertainty of extrapolation of cell biological findings from T cell hybridoma cells to primary CD8 T cells. Thus, increasing ligand density increases the frequency of responding B3Z cells but not the levels of reporter bGal expressed in responding cells. The response of pulse-activated B3Z cells is transient, allowing yet another parameter to estimate the duration of their response. Functional inhibition of Eps15 (as well as AP180 or dynamin-2) using transient transfection with DN constructs led to early enhancement of B3Z responses and slower TCR internalization, whereas inhibition of endolysosomal trafficking by DN-Rab7 (and DN-Rab21 and DN-Rab22) showed prolonged enhancement of B3Z responses and slower lysosomal delivery of internalized TCRs. Functional modulation of Rab5 showed a more complex phenotype as expected from its role in both endocytosis and endolysosomal trafficking. Some TCR-containing vesicles were associated with phospho-ZAP70, and this association was enhanced and persistent in DN-Rab7–expressing B3Z cells. These data further support the possibility that, upon T cell activation, some TCRs are internalized, perhaps with pMHC cargo, and continue to signal in endosomes until lysosomal delivery and degradation. Thus, alterations in vesicular trafficking, as shown recently for dynamin-2 (47), provide yet another potential source of heterogeneity in the modulation of T cell responses.

It must be kept in mind that the OT1–TCR–SIINFEKL–H-2Kb interactions are among the strongest known; as few as a hundred SIINFEKL–H-2Kb complexes per cell can generate a CD8 T cell response in vivo, and picomolar peptide concentrations can induce detectable cytolytic activity from OT1 effector cells (72). If effector CD8 T cells function on average at far lower peptide concentrations in vivo, they may lie in the low ligand density range of the present findings, and TCR internalization may not ordinarily be a relevant event in their functioning. Finally, the exceptionally high strength of the OT1–TCR–SIINFEKL–H-2Kb interactions raises the caveat that less potent TCR–peptide–MHC interactions may behave differently in quantitative or even in qualitative terms, adding further complexity to the understanding of antimicrobial CD8 T cell responses in vivo.

Nonetheless, our data show that ligand density or affinity is sensed at the population level by responding CD8 T cells based on nongenetic heterogeneity in activation thresholds. This heterogeneity is connected to variation in cellular metabolic status. Further, our data indicate that ligand density and affinity modulate the extent of TCR internalization, that internalized TCRs signal to enhance the rate of entry into the proliferation–differentiation program, and that signaling from internalized TCRs is terminated at endolysosomal fusion.

We thank Inderjit Singh and Dr. P. Nagarajan for help in animal breeding and maintenance and K. Rajesh Kumar for assistance in flow cytometric sorting.

This work was supported in part by grants from the Department of Biotechnology, Government of India (Grants BT/PR10954/BRB/10/625/2008 and BT/PR12849/MED/15/35/2009 to A.G.; Grants BT/PR-10284/MED/29/59/2007 and BT/PR14420/MED/29/213/2010 to V.B.; and Grant BT/PR-14592/BRB/10/858/2010 to S.R.), and from the Department of Science and Technology, Government of India (Grant SR/SO/HS-0005/2011 to V.B. and Grant SB/SO/HS/210/2013 to S.R.). The National Institute of Immunology is supported by the Department of Biotechnology, Government of India.

The online version of this article contains supplemental material.

Abbreviations used in this article:

bGal

β-galactosidase

BMDC

bone marrow–derived dendritic cell

CTV

Cell Trace Violet

DA

dominant-active

DN

dominant-negative

EdU

5-ethynyl-2′-deoxyuridine

eGFP

enhanced GFP

HPG

l-homopropargylglycine

2-NBDG

2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose

pMHC

peptide-MHC

pMHCI

peptide-MHC class I

pS6

phospho-S6

WT

wild-type.

1
Sykulev
Y.
,
Joo
M.
,
Vturina
I.
,
Tsomides
T. J.
,
Eisen
H. N.
.
1996
.
Evidence that a single peptide-MHC complex on a target cell can elicit a cytolytic T cell response.
Immunity
4
:
565
571
.
2
Smith-Garvin
J. E.
,
Koretzky
G. A.
,
Jordan
M. S.
.
2009
.
T cell activation.
Annu. Rev. Immunol.
27
:
591
619
.
3
van Heijst
J. W.
,
Gerlach
C.
,
Swart
E.
,
Sie
D.
,
Nunes-Alves
C.
,
Kerkhoven
R. M.
,
Arens
R.
,
Correia-Neves
M.
,
Schepers
K.
,
Schumacher
T. N.
.
2009
.
Recruitment of antigen-specific CD8+ T cells in response to infection is markedly efficient.
Science
325
:
1265
1269
.
4
Badovinac
V. P.
,
Porter
B. B.
,
Harty
J. T.
.
2002
.
Programmed contraction of CD8(+) T cells after infection.
Nat. Immunol.
3
:
619
626
.
5
Henrickson
S. E.
,
Mempel
T. R.
,
Mazo
I. B.
,
Liu
B.
,
Artyomov
M. N.
,
Zheng
H.
,
Peixoto
A.
,
Flynn
M. P.
,
Senman
B.
,
Junt
T.
, et al
.
2008
.
T cell sensing of antigen dose governs interactive behavior with dendritic cells and sets a threshold for T cell activation.
Nat. Immunol.
9
:
282
291
.
6
Valitutti
S.
,
Müller
S.
,
Dessing
M.
,
Lanzavecchia
A.
.
1996
.
Different responses are elicited in cytotoxic T lymphocytes by different levels of T cell receptor occupancy.
J. Exp. Med.
183
:
1917
1921
.
7
Auphan-Anezin
N.
,
Verdeil
G.
,
Schmitt-Verhulst
A. M.
.
2003
.
Distinct thresholds for CD8 T cell activation lead to functional heterogeneity: CD8 T cell priming can occur independently of cell division.
J. Immunol.
170
:
2442
2448
.
8
Busch
D. H.
,
Pilip
I. M.
,
Vijh
S.
,
Pamer
E. G.
.
1998
.
Coordinate regulation of complex T cell populations responding to bacterial infection.
Immunity
8
:
353
362
.
9
Zehn
D.
,
Lee
S. Y.
,
Bevan
M. J.
.
2009
.
Complete but curtailed T-cell response to very low-affinity antigen.
Nature
458
:
211
214
.
10
Valitutti
S.
,
Müller
S.
,
Cella
M.
,
Padovan
E.
,
Lanzavecchia
A.
.
1995
.
Serial triggering of many T-cell receptors by a few peptide-MHC complexes.
Nature
375
:
148
151
.
11
Müller
S.
,
Demotz
S.
,
Bulliard
C.
,
Valitutti
S.
.
1999
.
Kinetics and extent of protein tyrosine kinase activation in individual T cells upon antigenic stimulation.
Immunology
97
:
287
293
.
12
Au-Yeung
B. B.
,
Zikherman
J.
,
Mueller
J. L.
,
Ashouri
J. F.
,
Matloubian
M.
,
Cheng
D. A.
,
Chen
Y.
,
Shokat
K. M.
,
Weiss
A.
.
2014
.
A sharp T-cell antigen receptor signaling threshold for T-cell proliferation.
Proc. Natl. Acad. Sci. USA
111
:
E3679
E3688
.
13
Prlic
M.
,
Hernandez-Hoyos
G.
,
Bevan
M. J.
.
2006
.
Duration of the initial TCR stimulus controls the magnitude but not functionality of the CD8+ T cell response.
J. Exp. Med.
203
:
2135
2143
.
14
Blair
D. A.
,
Turner
D. L.
,
Bose
T. O.
,
Pham
Q. M.
,
Bouchard
K. R.
,
Williams
K. J.
,
McAleer
J. P.
,
Cauley
L. S.
,
Vella
A. T.
,
Lefrançois
L.
.
2011
.
Duration of antigen availability influences the expansion and memory differentiation of T cells.
J. Immunol.
187
:
2310
2321
.
15
Bullock
T. N.
,
Mullins
D. W.
,
Engelhard
V. H.
.
2003
.
Antigen density presented by dendritic cells in vivo differentially affects the number and avidity of primary, memory, and recall CD8+ T cells.
J. Immunol.
170
:
1822
1829
.
16
Bullock
T. N.
,
Colella
T. A.
,
Engelhard
V. H.
.
2000
.
The density of peptides displayed by dendritic cells affects immune responses to human tyrosinase and gp100 in HLA-A2 transgenic mice.
J. Immunol.
164
:
2354
2361
.
17
Mercado
R.
,
Vijh
S.
,
Allen
S. E.
,
Kerksiek
K.
,
Pilip
I. M.
,
Pamer
E. G.
.
2000
.
Early programming of T cell populations responding to bacterial infection.
J. Immunol.
165
:
6833
6839
.
18
Mempel
T. R.
,
Henrickson
S. E.
,
Von Andrian
U. H.
.
2004
.
T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases.
Nature
427
:
154
159
.
19
Alarcón
B.
,
Mestre
D.
,
Martínez-Martín
N.
.
2011
.
The immunological synapse: a cause or consequence of T-cell receptor triggering?
Immunology
133
:
420
425
.
20
Itoh
Y.
,
Germain
R. N.
.
1997
.
Single cell analysis reveals regulated hierarchical T cell antigen receptor signaling thresholds and intraclonal heterogeneity for individual cytokine responses of CD4+ T cells.
J. Exp. Med.
186
:
757
766
.
21
Bachmann
M. F.
,
Oxenius
A.
,
Speiser
D. E.
,
Mariathasan
S.
,
Hengartner
H.
,
Zinkernagel
R. M.
,
Ohashi
P. S.
.
1997
.
Peptide-induced T cell receptor down-regulation on naive T cells predicts agonist/partial agonist properties and strictly correlates with T cell activation.
Eur. J. Immunol.
27
:
2195
2203
.
22
Hwang
I.
,
Huang
J. F.
,
Kishimoto
H.
,
Brunmark
A.
,
Peterson
P. A.
,
Jackson
M. R.
,
Surh
C. D.
,
Cai
Z.
,
Sprent
J.
.
2000
.
T cells can use either T cell receptor or CD28 receptors to absorb and internalize cell surface molecules derived from antigen-presenting cells.
J. Exp. Med.
191
:
1137
1148
.
23
Kedl
R. M.
,
Schaefer
B. C.
,
Kappler
J. W.
,
Marrack
P.
.
2002
.
T cells down-modulate peptide-MHC complexes on APCs in vivo.
Nat. Immunol.
3
:
27
32
.
24
Fooksman
D. R.
,
Grönvall
G. K.
,
Tang
Q.
,
Edidin
M.
.
2006
.
Clustering class I MHC modulates sensitivity of T cell recognition.
J. Immunol.
176
:
6673
6680
.
25
Matko
J.
,
Bushkin
Y.
,
Wei
T.
,
Edidin
M.
.
1994
.
Clustering of class I HLA molecules on the surfaces of activated and transformed human cells.
J. Immunol.
152
:
3353
3360
.
26
Edidin
M.
2010
.
Class I MHC molecules as probes of membrane patchiness: from biophysical measurements to modulation of immune responses.
Immunol. Res.
47
:
265
272
.
27
Lu
X.
,
Gibbs
J. S.
,
Hickman
H. D.
,
David
A.
,
Dolan
B. P.
,
Jin
Y.
,
Kranz
D. M.
,
Bennink
J. R.
,
Yewdell
J. W.
,
Varma
R.
.
2012
.
Endogenous viral antigen processing generates peptide-specific MHC class I cell-surface clusters.
Proc. Natl. Acad. Sci. USA
109
:
15407
15412
.
28
Cebecauer
M.
,
Guillaume
P.
,
Mark
S.
,
Michielin
O.
,
Boucheron
N.
,
Bezard
M.
,
Meyer
B. H.
,
Segura
J. M.
,
Vogel
H.
,
Luescher
I. F.
.
2005
.
CD8+ cytotoxic T lymphocyte activation by soluble major histocompatibility complex-peptide dimers.
J. Biol. Chem.
280
:
23820
23828
.
29
Yachi
P. P.
,
Ampudia
J.
,
Gascoigne
N. R.
,
Zal
T.
.
2005
.
Nonstimulatory peptides contribute to antigen-induced CD8-T cell receptor interaction at the immunological synapse.
Nat. Immunol.
6
:
785
792
.
30
Anikeeva
N.
,
Lebedeva
T.
,
Clapp
A. R.
,
Goldman
E. R.
,
Dustin
M. L.
,
Mattoussi
H.
,
Sykulev
Y.
.
2006
.
Quantum dot/peptide-MHC biosensors reveal strong CD8-dependent cooperation between self and viral antigens that augment the T cell response.
Proc. Natl. Acad. Sci. USA
103
:
16846
16851
.
31
Anikeeva
N.
,
Gakamsky
D.
,
Schøller
J.
,
Sykulev
Y.
.
2012
.
Evidence that the density of self peptide-MHC ligands regulates T-cell receptor signaling.
PLoS One
7
:
e41466
.
32
Kalergis
A. M.
,
Boucheron
N.
,
Doucey
M. A.
,
Palmieri
E.
,
Goyarts
E. C.
,
Vegh
Z.
,
Luescher
I. F.
,
Nathenson
S. G.
.
2001
.
Efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex.
Nat. Immunol.
2
:
229
234
.
33
González
P. A.
,
Carreño
L. J.
,
Coombs
D.
,
Mora
J. E.
,
Palmieri
E.
,
Goldstein
B.
,
Nathenson
S. G.
,
Kalergis
A. M.
.
2005
.
T cell receptor binding kinetics required for T cell activation depend on the density of cognate ligand on the antigen-presenting cell.
Proc. Natl. Acad. Sci. USA
102
:
4824
4829
.
34
Karttunen
J.
,
Shastri
N.
.
1991
.
Measurement of ligand-induced activation in single viable T cells using the lacZ reporter gene.
Proc. Natl. Acad. Sci. USA
88
:
3972
3976
.
35
Inaba
K.
,
Inaba
M.
,
Romani
N.
,
Aya
H.
,
Deguchi
M.
,
Ikehara
S.
,
Muramatsu
S.
,
Steinman
R. M.
.
1992
.
Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor.
J. Exp. Med.
176
:
1693
1702
.
36
Chaudhry
A.
,
Das
S. R.
,
Jameel
S.
,
George
A.
,
Bal
V.
,
Mayor
S.
,
Rath
S.
.
2007
.
A two-pronged mechanism for HIV-1 Nef-mediated endocytosis of immune costimulatory molecules CD80 and CD86.
Cell Host Microbe
1
:
37
49
.
37
Ghosh
R. N.
,
Gelman
D. L.
,
Maxfield
F. R.
.
1994
.
Quantification of low density lipoprotein and transferrin endocytic sorting HEp2 cells using confocal microscopy.
J. Cell Sci.
107
:
2177
2189
.
38
Hogquist
K. A.
,
Jameson
S. C.
,
Heath
W. R.
,
Howard
J. L.
,
Bevan
M. J.
,
Carbone
F. R.
.
1994
.
T cell receptor antagonist peptides induce positive selection.
Cell
76
:
17
27
.
39
Porgador
A.
,
Yewdell
J. W.
,
Deng
Y.
,
Bennink
J. R.
,
Germain
R. N.
.
1997
.
Localization, quantitation, and in situ detection of specific peptide-MHC class I complexes using a monoclonal antibody.
Immunity
6
:
715
726
.
40
van Stipdonk
M. J.
,
Lemmens
E. E.
,
Schoenberger
S. P.
.
2001
.
Naïve CTLs require a single brief period of antigenic stimulation for clonal expansion and differentiation.
Nat. Immunol.
2
:
423
429
.
41
Kaech
S. M.
,
Ahmed
R.
.
2001
.
Memory CD8+ T cell differentiation: initial antigen encounter triggers a developmental program in naïve cells.
Nat. Immunol.
2
:
415
422
.
42
Altan-Bonnet
G.
,
Germain
R. N.
.
2005
.
Modeling T cell antigen discrimination based on feedback control of digital ERK responses.
PLoS Biol.
3
:
e356
.
43
Das
J.
,
Ho
M.
,
Zikherman
J.
,
Govern
C.
,
Yang
M.
,
Weiss
A.
,
Chakraborty
A. K.
,
Roose
J. P.
.
2009
.
Digital signaling and hysteresis characterize ras activation in lymphoid cells.
Cell
136
:
337
351
.
44
Wolfraim
L. A.
,
Letterio
J. J.
.
2005
.
Cutting edge: p27Kip1 deficiency reduces the requirement for CD28-mediated costimulation in naive CD8+ but not CD4+ T lymphocytes.
J. Immunol.
174
:
2481
2484
.
45
Kay
J. E.
,
Ahern
T.
,
Atkins
M.
.
1971
.
Control of protein synthesis during the activation of lymphocytes by phytohaemagglutinin.
Biochim. Biophys. Acta
247
:
322
334
.
46
Macintyre
A. N.
,
Gerriets
V. A.
,
Nichols
A. G.
,
Michalek
R. D.
,
Rudolph
M. C.
,
Deoliveira
D.
,
Anderson
S. M.
,
Abel
E. D.
,
Chen
B. J.
,
Hale
L. P.
,
Rathmell
J. C.
.
2014
.
The glucose transporter Glut1 is selectively essential for CD4 T cell activation and effector function.
Cell Metab.
20
:
61
72
.
47
Willinger
T.
,
Staron
M.
,
Ferguson
S. M.
,
De Camilli
P.
,
Flavell
R. A.
.
2015
.
Dynamin 2-dependent endocytosis sustains T-cell receptor signaling and drives metabolic reprogramming in T lymphocytes.
Proc. Natl. Acad. Sci. USA
112
:
4423
4428
.
48
Sykulev
Y.
,
Cohen
R. J.
,
Eisen
H. N.
.
1995
.
The law of mass action governs antigen-stimulated cytolytic activity of CD8+ cytotoxic T lymphocytes.
Proc. Natl. Acad. Sci. USA
92
:
11990
11992
.
49
Sprent
J.
1995
.
Antigen-presenting cells. Professionals and amateurs.
Curr. Biol.
5
:
1095
1097
.
50
Kündig
T. M.
,
Bachmann
M. F.
,
DiPaolo
C.
,
Simard
J. J.
,
Battegay
M.
,
Lother
H.
,
Gessner
A.
,
Kühlcke
K.
,
Ohashi
P. S.
,
Hengartner
H.
, et al
.
1995
.
Fibroblasts as efficient antigen-presenting cells in lymphoid organs.
Science
268
:
1343
1347
.
51
Nickoloff
B. J.
,
Turka
L. A.
.
1994
.
Immunological functions of non-professional antigen-presenting cells: new insights from studies of T-cell interactions with keratinocytes.
Immunol. Today
15
:
464
469
.
52
Steinman
R. M.
1991
.
The dendritic cell system and its role in immunogenicity.
Annu. Rev. Immunol.
9
:
271
296
.
53
Wang
R.
,
Dillon
C. P.
,
Shi
L. Z.
,
Milasta
S.
,
Carter
R.
,
Finkelstein
D.
,
McCormick
L. L.
,
Fitzgerald
P.
,
Chi
H.
,
Munger
J.
,
Green
D. R.
.
2011
.
The transcription factor Myc controls metabolic reprogramming upon T lymphocyte activation.
Immunity
35
:
871
882
.
54
Marko
A. J.
,
Miller
R. A.
,
Kelman
A.
,
Frauwirth
K. A.
.
2010
.
Induction of glucose metabolism in stimulated T lymphocytes is regulated by mitogen-activated protein kinase signaling.
PLoS One
5
:
e15425
.
55
Ozbudak
E. M.
,
Thattai
M.
,
Kurtser
I.
,
Grossman
A. D.
,
van Oudenaarden
A.
.
2002
.
Regulation of noise in the expression of a single gene.
Nat. Genet.
31
:
69
73
.
56
Feinerman
O.
,
Veiga
J.
,
Dorfman
J. R.
,
Germain
R. N.
,
Altan-Bonnet
G.
.
2008
.
Variability and robustness in T cell activation from regulated heterogeneity in protein levels.
Science
321
:
1081
1084
.
57
Rane
S.
,
Das
R.
,
Ranganathan
V.
,
Prabhu
S.
,
Das
A.
,
Mattoo
H.
,
Durdik
J. M.
,
George
A.
,
Rath
S.
,
Bal
V.
.
2014
.
Peripheral residence of naïve CD4 T cells induces MHC class II-dependent alterations in phenotype and function.
BMC Biol.
12
:
106
.
58
Smith
K.
,
Seddon
B.
,
Purbhoo
M. A.
,
Zamoyska
R.
,
Fisher
A. G.
,
Merkenschlager
M.
.
2001
.
Sensory adaptation in naive peripheral CD4 T cells.
J. Exp. Med.
194
:
1253
1261
.
59
Ferrell
J. E.
 Jr.
,
Machleder
E. M.
.
1998
.
The biochemical basis of an all-or-none cell fate switch in Xenopus oocytes.
Science
280
:
895
898
.
60
Huang
C. Y.
,
Ferrell
J. E.
 Jr.
1996
.
Ultrasensitivity in the mitogen-activated protein kinase cascade.
Proc. Natl. Acad. Sci. USA
93
:
10078
10083
.
61
Lee
K. H.
,
Dinner
A. R.
,
Tu
C.
,
Campi
G.
,
Raychaudhuri
S.
,
Varma
R.
,
Sims
T. N.
,
Burack
W. R.
,
Wu
H.
,
Wang
J.
, et al
.
2003
.
The immunological synapse balances T cell receptor signaling and degradation.
Science
302
:
1218
1222
.
62
Cemerski
S.
,
Das
J.
,
Giurisato
E.
,
Markiewicz
M. A.
,
Allen
P. M.
,
Chakraborty
A. K.
,
Shaw
A. S.
.
2008
.
The balance between T cell receptor signaling and degradation at the center of the immunological synapse is determined by antigen quality.
Immunity
29
:
414
422
.
63
Naramura
M.
,
Jang
I. K.
,
Kole
H.
,
Huang
F.
,
Haines
D.
,
Gu
H.
.
2002
.
c-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation.
Nat. Immunol.
3
:
1192
1199
.
64
Valitutti
S.
,
Müller
S.
,
Salio
M.
,
Lanzavecchia
A.
.
1997
.
Degradation of T cell receptor (TCR)-CD3-zeta complexes after antigenic stimulation.
J. Exp. Med.
185
:
1859
1864
.
65
Rosette
C.
,
Werlen
G.
,
Daniels
M. A.
,
Holman
P. O.
,
Alam
S. M.
,
Travers
P. J.
,
Gascoigne
N. R.
,
Palmer
E.
,
Jameson
S. C.
.
2001
.
The impact of duration versus extent of TCR occupancy on T cell activation: a revision of the kinetic proofreading model.
Immunity
15
:
59
70
.
66
Luton
F.
,
Legendre
V.
,
Gorvel
J. P.
,
Schmitt-Verhulst
A. M.
,
Boyer
C.
.
1997
.
Tyrosine and serine protein kinase activities associated with ligand-induced internalized TCR/CD3 complexes.
J. Immunol.
158
:
3140
3147
.
67
Chaturvedi
A.
,
Martz
R.
,
Dorward
D.
,
Waisberg
M.
,
Pierce
S. K.
.
2011
.
Endocytosed BCRs sequentially regulate MAPK and Akt signaling pathways from intracellular compartments.
Nat. Immunol.
12
:
1119
1126
.
68
Vieira
A. V.
,
Lamaze
C.
,
Schmid
S. L.
.
1996
.
Control of EGF receptor signaling by clathrin-mediated endocytosis.
Science
274
:
2086
2089
.
69
Osborne
D. G.
,
Wetzel
S. A.
.
2012
.
Trogocytosis results in sustained intracellular signaling in CD4(+) T cells.
J. Immunol.
189
:
4728
4739
.
70
Kagan
J. C.
,
Su
T.
,
Horng
T.
,
Chow
A.
,
Akira
S.
,
Medzhitov
R.
.
2008
.
TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta.
Nat. Immunol.
9
:
361
368
.
71
Feng
Y.
,
Press
B.
,
Wandinger-Ness
A.
.
1995
.
Rab 7: an important regulator of late endocytic membrane traffic.
J. Cell Biol.
131
:
1435
1452
.
72
Rötzschke
O.
,
Falk
K.
,
Stevanović
S.
,
Jung
G.
,
Walden
P.
,
Rammensee
H. G.
.
1991
.
Exact prediction of a natural T cell epitope.
Eur. J. Immunol.
21
:
2891
2894
.

The authors have no financial conflicts of interest.

Supplementary data