TCR Signal Strength and Duration In Primary Mouse CD8+ T Cell Activation and Differentiation: A Standardized In Vitro Approach

Summary

This protocol describes a systematic method to activate primary mouse naive CD8⁺ T cells using a graded TCR stimulation approach, enabling comprehensive analysis of proliferation, activation, and cytokine production of CD8⁺ T cells across different TCR signal intensities.

Abstract

T cell receptor (TCR) signal intensity and duration are critical determinants of CD8⁺ T cell activation, proliferation, and functional differentiation. However, standardized in vitro models that systematically compare how varying TCR signal inputs shape T cell fate remain limited. This study presents a reproducible method for activating primary mouse T cells in vitro using plate-bound anti-CD3 and soluble anti-CD28, supplemented with interleukin-2 (IL-2), and examines how modulating anti-CD3 concentration (signal strength) and stimulation time (signal duration) influences activation outcomes of T cells. We detail a protocol for isolating naive T cells from C57BL/6 mice, followed by stimulation across a range of anti-CD3 concentrations (0.1–10 µg/mL) and durations (6–48 h). We conducted flow cytometric analysis to assess early TCR-induced signaling via intracellular phosphorylated S6 ribosomal protein (p-S6) as a readout of mechanistic target of rapamycin complex 1 (mTORC1) activity, and then evaluated downstream events including surface activation markers (CD69, CD25, ICOS, PD-1), cellular proliferation via violet proliferation-tracking dye (CTV) dilution, and effector molecule expression (Granzyme B, TNF-α) as indicators of functional differentiation. The method provides a clear, stepwise visual guide for executing reproducible T cell stimulations, enabling researchers to dissect how specific TCR signal parameters direct T cell fate decisions. This approach not only supports mechanistic studies of T cell activation and differentiation but also offers a platform for optimizing T cell conditioning for adoptive immunotherapy and in vitro priming assays. By integrating video demonstration with detailed protocol narration, this protocol will enhance methodological transparency and reproducibility in T cell immunology research.

Introduction

T cells are indispensable for protective immune responses against pathogens and tumors. Naive T cells are actively maintained in a quiescent state to preserve their long-term survival and persistence¹. Upon antigen stimulation, T cells exit quiescence, undergo clonal expansion and effector differentiation². T cell receptor (TCR) signaling serves as the core process for initiating and regulating adaptive immune responses³. Upon specific recognition of peptide-major histocompatibility complex (pMHC) molecules presented on the surface of antigen-presenting cells (APCs), a cascade of signaling events is triggered, thereby driving clonal expansion and functional differentiation of T cells^4,5,6.

The intensity of TCR stimulation, determined by key factors including antigen affinity, avidity, and concentration, plays a critical role in governing the magnitude of T cell activation, the acquisition of effector functions, and the differentiation trajectory toward distinct effector or memory T cell subsets^7,8,9. Current in vitro models for investigating TCR-dependent T cell activation predominantly employ plate-bound or bead-conjugated anti-CD3 and anti-CD28 antibodies, which provide both TCR ligation and co-stimulatory signals simultaneously^10,11. Although such systems are widely used, they typically utilize only a single, often saturating concentration of anti-CD3 antibody, permitting merely an all-or-nothing binary comparison of T cell activation, rather than capturing the dynamic range of TCR signal strengths achievable with a titrated gradient of stimuli^12,13.

The protocol described herein provides a standardized and reproducible in vitro system to systematically evaluate how graded TCR signal strengths modulate activation, proliferation, and functional phenotypes of primary murine CD8⁺ T cells, using titrated anti-CD3 gradients (0.1–10 µg/mL) and a constant anti-CD28 (1 µg/mL). This approach centers on multiparameter flow cytometry that captures both upstream signaling—via intracellular phosphorylated S6 ribosomal protein (p-S6) as a readout of mechanistic target of rapamycin complex 1 (mTORC1) activity—and downstream cellular outcomes, including proliferation tracked by CTV dilution, activation surface markers (CD69, CD25, inducible T-cell costimulatory [ICOS], programmed cell death protein 1 [PD-1]), and effector molecule expression (Granzyme B, tumor necrosis factor-alpha [TNF-α]), over a time course from 6 to 48 h.

Our protocol employs a gradient concentration of plate-bound anti-CD3 antibodies to precisely modulate stimulation intensity, thereby mimicking the differential TCR binding avidity observed under physiological conditions. The method achieves high efficiency in TCR crosslinking and robust activation of primary resting T cells. The standardized coating procedure ensures uniform antibody distribution on the plate surface, leading to consistent stimulation across cells within the same batch and excellent experimental reproducibility. This format provides a cost-effective and operationally simple approach for T cell activation.

However, certain limitations of this approach should be acknowledged. Stimulation mediated by anti-CD3/CD28 antibodies differs markedly from the in vivo physiological context. Although experimental methods that use only agonistic anti-CD3 and anti-CD28 antibodies to assess T cell activation can provide effective readouts of activation, they fail to fully recapitulate the interactions between antigen-presenting cells and T cells, as well as the co-stimulatory microenvironment that occur under physiological conditions¹⁴.

Despite these limitations, this protocol offers a systematic and reproducible platform that serves as a valuable and accessible tool for investigating TCR signal strength-dependent T cell responses and can complement more complex antigen-specific models.

Protocol

All animal experiments in this study were approved by the Animal Care and Use Committee of Third Military Medical University.

1. Preparation of reagents and plates

1. Prepare plate-bound anti-CD3 in sterile PBS at 0.1, 0.5, 1, 5, and 10 µg/mL.
2. Add 50 µL of the anti-CD3 coating solution per well into flat-bottom 96-well plates.
3. Seal plates with sealing film and incubate overnight at 4 °C.
4. Aspirate the coating solution and wash each well twice with 200 µL of sterile PBS before adding cells.

2. Isolation of naive CD8⁺ T cells from mouse spleen

1. Select 6–8-week-old male C57BL/6 mice (purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) and euthanize by CO₂ inhalation followed by cervical dislocation. Mice are housed under specific pathogen-free conditions with a 12-h light/dark cycle and free access to food and water. A minimum of 3 mice are used per independent experiment.
2. Carefully incise the peritoneum with surgical scissors to expose the spleen, grasp and harvest the spleen using forceps, and place it into a collection dish containing pre-chilled RPMI 1640 supplemented with 2% fetal bovine serum(FBS) (R2). Keep the dish on ice until dissociation.
   NOTE: Collect all animal carcasses and cell culture waste in designated biohazard bags and autoclave them at 121 °C for 20 min before disposal.
3. Remove the spleen from the collection dish and transfer it into a 60 mm dish containing 3 mL of 1 × ammonium-chloride-potassium (ACK) lysis buffer.
4. Prepare a single-cell suspension by gently mashing the spleen through a 70 µm cell strainer using the plunger of a 3 mL syringe.
5. Incubate the suspension for 1 min at room temperature(RT), then add 3 mL of pre-chilled R2 medium to neutralize the ammonium-chloride-potassium (ACK) lysis buffer.
6. Transfer the cell suspension to a 15 mL conical tube and centrifuge at 500 × g for 5 min at 4 °C.
7. After centrifugation, aspirate the supernatant and resuspend the pellet in 10 mL of flow cytometry staining buffer (PBS with 2% FBS).
8. Filter the suspension through a 70 µm cell strainer into a new 15 mL conical tube, then centrifuge again at 500 × g for 5 min at 4 °C.
   NOTE: Sodium azide should be omitted from flow cytometry staining buffer to preserve cell viability for functional assays. All samples should be kept at 4 °C and analyzed immediately.
9. Aspirate the supernatant and resuspend the cell pellet in 200 µL of biotinylated antibody cocktail per mouse spleen (containing anti-CD16/32 (Fc block), anti-TER-119, anti-CD19, anti-CD4, anti-B220, anti-NK1.1, anti-Ly-6G, anti-CD11b, anti-CD11c, anti-CD49b, anti-CD44 and anti-F4/80).
10. Incubate on ice for 25 min with gentle inversion of the tube every 5 min to ensure thorough mixing.
11. Wash the cells with a large volume of flow cytometry staining buffer, centrifuge at 500 × g for 5 min and discard the supernatant.
12. Resuspend the pellet in the same volume of flow cytometry staining buffer as used for biotinylated antibody cocktail, add streptavidin-coated magnetic beads at half that volume, and incubate on ice for 15 min with gentle inversion of the tube every 5 min to ensure thorough mixing.
13. Briefly centrifuge the tube, place it on a magnetic stand for 2–3 min, and carefully transfer the supernatant (enriched naive CD8⁺ T cells) to a new 15 mL conical tube.
14. Wash the collected cells once with flow cytometry staining buffer, centrifuge again at 500 × g for 5 min.
15. Discard the supernatant and resuspend the pellet in RPMI 1640 supplemented with 10% FBS (R10) for downstream applications.

3. Violet proliferation-tracking dye (CTV) labeling for proliferation tracking

1. Prepare 2 × fresh CTV working solution (10 µM) immediately before use. Fully mix the 5 mM CTV stock solution (in dimethyl sulfoxide [DMSO]) by vortexing and further dilute it with sterile PBS to obtain a final concentration of 10 µM.
   CAUTION: Handle CTV stock solution (prepared in DMSO) with nitrile gloves in a fume hood, as DMSO facilitates skin absorption of toxic compounds. Avoid inhalation and skin contact.
2. Resuspend half of the isolated naive CD8⁺ T cells in pre-chilled PBS at a density of 8 × 10⁷ cells/mL.
3. Rapidly add an equal volume of 2 × CTV working solution (10 µM) to the cell suspension and mix well by pipetting, yielding a final CTV concentration of 5 µM.
4. Incubate the cells at RT in the dark for 20 min, with gentle inversion of the tubes every 5 min to ensure thorough mixing.
5. Add 5 volumes of culture medium (containing at least 10% FBS) to the cells and incubate for 5 min on ice to remove any free dye.
6. Centrifuge cells at 500 × g for 5 min and resuspend the pellet in freshly pre-warmed complete medium R10 supplemented with anti-CD28 (final concentration: 1 µg/mL) and IL-2 (final concentration: 10 ng/mL).
7. Add 250 µL of cell suspension (containing 6 × 10⁵cells) to each well of the pre-coated 96-well plates.
8. Culture cells at 37 °C with 5% CO₂ for up to 48 h.

4. CTV detection by flow cytometry

1. Collect cells into a 96-well round-bottom plate at 6, 36, and 48 h. Centrifuge at 500 × g for 5 min and wash twice with flow cytometry staining buffer.
2. For surface staining, incubate cells with 50 µL of an antibody cocktail containing anti-CD8, fixable viability dye, and anti-CD44 for 30 min on ice in the dark.
   NOTE: Collect all supernatants containing fluorophore-conjugated antibodies in designated waste containers supplemented with 0.5% sodium hypochlorite for decontamination and discard them according to institutional biohazardous waste protocols.
3. Wash cells twice with flow cytometry staining buffer and resuspend in 200 µL of flow cytometry staining buffer for flow cytometer acquisition.
4. Acquire samples on a flow cytometer equipped with three lasers. Prepare single-color control tubes using stained cells to set compensation parameters.
5. Apply the following gating strategy: exclude debris by forward-scatter (FSC) versus side-scatter (SSC) gating, eliminate doublets by singlet gating, and select viable (fixable viability dye-negative) CD8⁺ T cells for analysis.
6. Collect at least 1 × 10⁴ gated CD8⁺ T cell events per sample. Analyze the data using the flow cytometry analysis software and calculate the proportion of cells that have undergone one or more divisions using the proliferation analysis module.

5. Surface and intracellular staining for activation and signaling

1. Resuspend another half of naive CD8⁺ T cells in R10 supplemented with anti-CD28 at 1 µg/mL and IL-2 at 10 ng/mL at 2.4 × 10⁶ cells/mL.
2. Add 250 µL of cell suspension (6 × 10⁵cells) to each well of the pre-coated 96-well plates.
3. Culture cells at 37 °C with 5% CO₂ for up to 48 h.
4. At indicated time points (6, 10, 12, 24, and 36 h), collect cells into a 96-well round-bottom plate.
   NOTE: 6 h captures early TCR signaling (phospho-S6) and CD69; the latter is additionally assessed at 12, 24, and 36 h to capture its full kinetics. CD25, PD-1, and ICOS are monitored at 10, 12, 24, and 36 h; the 48 h time point is reserved for proliferation (Section 4) and cytokine detection (Section 6).
5. Centrifuge at 500 × g for 1 min and resuspend cell pellet in 200 µL of flow cytometry staining buffer.
6. Centrifuge at 500 × g for 1 min and aspirate supernatant. Wash cells twice with 200 µL of flow cytometry staining buffer.
7. For surface staining, incubate cells with 50 µL of an antibody cocktail containing anti-CD69, anti-CD25, anti-CD8, anti-PD-1, anti-ICOS, and fixable viability dye for 30 min on ice in the dark.
8. Wash cells three times with flow cytometry staining buffer to remove unbound antibodies.
9. Resuspend the cell pellet in 200 µL of 1% paraformaldehyde, and fix cells at RT in dark for 15 min.
   CAUTION: Perform all steps involving paraformaldehyde in a fume hood to prevent inhalation of vapors. Collect all liquid waste containing fixative in a separate container marked for hazardous chemical disposal.
10. Centrifuge at 500 × g for 1 min and wash once with flow cytometry staining buffer.
11. Pre-warm 5 × lyse/fix buffer at RT and prepare 1 × working solution with distilled water.
12. Add 200 µL working solution (1 × lyse/fix buffer) into each well, incubate cells at RT in dark for 30 min.
13. Wash twice with 1 × permeabilization/wash buffer for phospho-epitope staining. Resuspend the pellet with 1 × permeabilization/wash buffer for phospho-epitope staining and incubate at RT in dark for 10 min.
14. Centrifuge at 500 × g for 1 min and discard supernatants.
15. For intracellular staining, incubate cells with 50 µL of an antibody cocktail containing anti-p-S6 for 60 min at RT in the dark.
16. Wash cells three times with 1 × permeabilization/wash buffer for phospho-epitope staining.
17. For secondary antibody staining, incubate cells with 50 µL of an antibody cocktail containing anti-rabbit secondary antibody for 30 min on ice in the dark.
18. Wash cells three times with 1 × permeabilization/wash buffer for phospho-epitope staining and wash once with flow cytometry staining buffer.
19. Resuspend the cell pellet in 200 µL of flow cytometry staining buffer and acquire samples on the same flow cytometer.
20. Apply the same gating strategy as described in Section 4.5 and collect at least 1 × 10⁴ CD8⁺ T cells per sample.
21. Analyze the median fluorescence intensity (MFI) for each marker using flow cytometry analysis software.

6. Intracellular cytokine blocking and detection

1. 5 h before harvest, add 100 µL per well of fresh R10 supplemented with PMA (50 ng/mL), ionomycin (1 µg/mL), protein transport inhibitors (monensin) (1:1000) and protein transport inhibitors (brefeldin A–based)(1:1000). Gently mix and return the plates to the 37 °C incubator to block cytokine secretion and allow intracellular accumulation.
2. At the desired time point, collect cells into a 96-well round-bottom plate.
3. Centrifuge at 500 × g for 1 min and resuspend cell pellet in 200 µL of flow cytometry staining buffer.
4. Centrifuge at 500 × g for 1 min and aspirate supernatant. Wash twice with 200 µL of flow cytometry staining buffer.
5. For surface marker staining, incubate cells with 50 µL of an antibody cocktail containing anti-CD44, anti-CD8 and fixable viability dye for 30 min on ice in the dark.
6. Wash three times with flow cytometry staining buffer to remove unbound antibodies.
7. Permeabilize the cells by adding 200 µL of fixation and permeabilization solution and incubate for 30 min on ice in the dark.
   CAUTION: Perform all steps involving Fixation/Permeabilization Solution (containing paraformaldehyde) in a fume hood to prevent inhalation of vapors. Collect all liquid waste containing fixative in a separate container marked for hazardous chemical disposal.
8. Centrifuge at 500 × g for 1 min and discard the supernatant into hazardous chemical waste.
9. Wash cells three times with 1 × permeabilization/wash buffer.
10. For intracellular cytokine staining, incubate with 50 µL of an antibody cocktail containing anti-TNF-α and anti-Granzyme B for 30 min on ice in the dark.
11. Wash cells three times with 1 × permeabilization/wash buffer to remove unbound antibodies and perform one final wash with flow cytometry staining buffer.
12. Resuspend the cell pellet in 200 µL of flow cytometry staining buffer and acquire samples on the same flow cytometer.
13. Apply the same gating strategy as described in Section 4.5 and collect at least 1 × 10⁴ CD8⁺ T cells per sample. Analyze the percent positivity and MFI for each marker using flow cytometry analysis software.

Results

To investigate the effects of different anti-CD3 stimulation intensities on T cell activation, we isolated murine splenocytes and purified CD8⁺ T cells for in vitro culture and then performed the T cell activation assays and flow cytometric analysis. For data analysis, lymphocytes were first gated using a combination of FSC-A and SSC-A, followed by gating of singlets via FSC-H and FSC-W, and then SSC-H with SSC-W. Live CD8⁺ T cells were further gated using CD8 combined with fixable viability dye for downstream analysis (Figure 1A).

To validate that the plate-bound antibody stimulation system effectively activates T cells in vitro, we examined the phosphorylation of ribosomal protein S6 (p-S6), a well-established downstream readout of mTORC1 activity and a point of convergence for PI3K-mTOR and MEK-ERK MAPK pathways downstream of TCR signaling^15,16. Consistent with these findings, we observed a dose-dependent increase in p-S6 levels at 6 h post-stimulation across increasing anti-CD3 concentrations (0.1–10 µg/mL) (Figure 1B), confirming that the plate-bound anti-CD3 system activates the TCR-mTORC1-S6 axis in a signal strength-dependent manner. This early phosphorylation event is consistent with engagement of mTORC1-dependent metabolic pathways that support subsequent T cell proliferation and effector differentiation.

Cell proliferation was assessed by CTV dilution flow cytometry following TCR stimulation with graded anti-CD3 concentrations (0.1–10 µg/mL) at three time points post-stimulation: 6, 36, and 48 h. At the early 6 h signaling time point, only minimal proliferative dilution was observed across all stimulation conditions. By 36 h, the low-dose stimulation group (0.1 µg/mL) still failed to induce appreciable clonal expansion, whereas intermediate- and high-dose groups exhibited well-resolved CTV dilution peaks (5 and 10 µg/mL) , indicating the onset of cell cycle progression. At 48 h, the differential proliferative phenotypes across stimulation concentrations were most pronounced. Weak TCR stimulation (0.1 µg/mL) yielded only 1.25% proliferating cells and a single undiluted CTV peak, indicating suboptimal activation. Intermediate stimulation (1–5 µg/mL) induced three resolved division peaks with proliferating fractions increasing from 70.8% to 84.0%. Strong stimulation (10 µg/mL) reached the highest proliferating fraction (89.5%), displaying the maximal number of division generations and demonstrating detection of proliferation saturation (Figure 2).

We next examined the expression of early activation markers CD69 and CD25. Notably, the present study revealed a rapid and dose-dependent upregulation of CD69 expression, based on the median fluorescence intensity (MFI). Under intermediate and strong stimulation (5 and 10 µg/mL), CD69 was rapidly elevated at 24 h, followed by a gradual decline between 24 and 36 h. However, under weak stimulation (0.1, 0.5, and 1 µg/mL), CD69 was progressively elevated during the whole stimulation window. The above kinetic expression profile of CD69 is consistent with its role as an early T cell activation marker (Figure 3A)¹⁷. Interestingly, we observed that CD25 expression exhibited a progressive increase in a dose- and time-dependent manner (Figure 3B). Consistent with previous research, the upregulation of CD25 was slower than that of CD69, especially upon optimal stimulation^18,19.

To further investigate the regulatory effect of TCR stimulation strength on the expression of inhibitory receptors and co-stimulatory molecules in CD8⁺ T cells, we examined the expression levels of PD-1 and ICOS. Inducible expression of PD-1 relies on high-intensity TCR stimulation. In the low-intensity stimulation group (0.5–1 µg/mL), PD-1 was expressed at a low level throughout the entire stimulation period. In contrast, in the medium-to-high intensity stimulation group (5–10 µg/mL), PD-1 expression increased gradually from 10 h and peaked at 36 h (Figure 4A). The expression of ICOS also rose accordingly with increasing stimulation strength and reached its maximum level at 5–10 µg/mL at 36 h (Figure 4B). These results suggest that high-intensity TCR stimulation can significantly upregulate the expression levels of PD-1 and ICOS.

Finally, we examined the functional capacity of stimulated T cells upon PMA and ionomycin restimulation at 24 h and 48 h post-stimulation. At 24 h, we quantified the production of TNF-α and Granzyme B and observed that the frequencies of TNF-α⁺, Granzyme B⁺, and TNF-α⁺Granzyme B⁺ cells were gradually elevated in an anti-CD3 dose-dependent manner (Figure 5A, B). Consistently, the MFI of TNF-α and Granzyme B also showed a concentration-dependent increase and reached a maximum under high-intensity stimulation (10 µg/mL) (Figure 5C), reflecting dose-dependent early effector differentiation driven by graded TCR signal strength. At 48 h, however, this pattern markedly reversed. Higher anti-CD3 concentrations significantly reduced the percentages of TNF-α⁺Granzyme B⁺ cells and downregulated the expression levels of effector molecules on a per-cell basis.

All data shown are representative of 3 independent experiments. Statistical analyses were performed using one-way or two-way ANOVA with Tukey’s post-hoc test; data are represented as mean ± SEM. Cell viability consistently exceeded 90%, and a minimum of 1 × 10⁴live CD8⁺ T cell events were acquired per sample.

Figure 1: In vitro activation and flow cytometric analysis of CD8⁺ T cells. (A) Purified CD8⁺ T cells from mouse splenocytes were stimulated in vitro with varying concentrations of anti-CD3 (0.1–10 µg/mL) combined with a fixed concentration of anti-CD28 (1 µg/mL) for 6 to 48 h, followed by flow cytometric analysis of cellular activation. Cellular debris was excluded by gating on forward scatter (FSC) and side scatter (SSC), doublets were excluded via singlet gating, and fixable viability dye-negative, CD8⁺ T cells were finally gated for downstream analyses. (B) Phosphorylation of ribosomal protein S6 (p-S6) in CD8⁺ T cells at 6 h post-stimulation with indicated concentrations of anti-CD3 (0.1–10 µg/mL) plus constant anti-CD28 (1 µg/mL). Representative histogram (left) and MFI summary (right). Flow cytometry plots are representative of 3 independent experiments. Data show mean ± SEM. Statistics: repeated-measures one-way ANOVA with Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. Please click here to view a larger version of this figure.

Figure 2: Violet proliferation-tracking dye (CTV)-based proliferation analysis of primary mouse CD8⁺ T cells under graded TCR stimulation. CD8⁺ T cells labeled with CTV and stimulated in vitro with anti-CD3 at gradient concentrations of 0.1–10 µg/mL for 6 to 48 h. Cell proliferation was assessed based on the dilution and attenuation of CTV fluorescence in CD8⁺ T cells by flow cytometric analysis. Histograms show the CTV fluorescence distribution of each concentration group, with the percentage of cells undergoing one or more divisions labeled. Flow cytometry plots are representative of 3 independent experiments. Please click here to view a larger version of this figure.

Figure 3: Expression kinetics of early activation markers CD69 and CD25 on CD8⁺ T cells following anti-CD3 stimulation. Naive CD8⁺ T cells were stimulated with plate-bound anti-CD3 at the indicated concentrations (0.1–10 µg/mL) plus constant anti-CD28 (1 µg/mL). CD69 was measured at 6, 12, 24, and 36 h; CD25 at 10, 12, 24, and 36 h and analyzed by flow cytometry. Left panels show representative histograms at the indicated time points; right panels present the dynamic changes in MFI over time. (A) CD69. (B) CD25. Flow cytometry plots are representative of 3 independent experiments. Data show mean ± SEM. Statistics: repeated-measures two-way ANOVA with Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. Please click here to view a larger version of this figure.

Figure 4: Expression kinetics of the inhibitory receptor PD-1 and co-stimulatory molecule ICOS on CD8⁺ T cells following anti- CD3 stimulation. Naive CD8⁺ T cells were stimulated with plate-bound anti-CD3 at the indicated concentrations (0.1–10 µg/mL) plus constant anti-CD28 (1 µg/mL). Cells were harvested at 10, 12, 24, and 36 h and analyzed by flow cytometry. Left panels show representative histograms at the indicated time points; right panels present the dynamic changes in MFI over time. (A) PD-1. (B) ICOS. Flow cytometry plots are representative of 3 independent experiments. Data show mean ± SEM. Statistics: repeated-measures two-way ANOVA with Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. Please click here to view a larger version of this figure.

Figure 5: Intracellular cytokine production by CD8⁺ T cells depends on TCR strength. Naive CD8⁺ T cells were stimulated with increasing concentrations of anti-CD3 (0.1–10 µg/mL) plus constant anti-CD28 (1 µg/mL). At 24 h and 48 h post-stimulation, cells were harvested following a 5-h incubation with protein transport inhibitors (monensin and brefeldin A) in the presence of PMA and ionomycin to block cytokine secretion and allow intracellular accumulation. (A) Contour plots show the frequencies of TNF-α- and Granzyme B-positive cells at each indicated concentration and time point. Unstimulated cells served as negative control. (B) The proportions of TNF-α⁺, Granzyme B⁺, and TNF-α⁺ Granzyme B⁺ cells. (C) The MFI of TNF-α and Granzyme B. Flow cytometry plots are representative of 3 independent experiments. Data show mean ± SEM. Statistics: repeated-measures two-way ANOVA with Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant. Please click here to view a larger version of this figure.

Discussion

This protocol provides a systematic and reproducible method for studying TCR signal strength-dependent T cell responses. The graded anti-CD3 coating strategy allows precise control over stimulation intensity, enabling researchers to dissect the quantitative relationship between TCR input and downstream functional outcomes. Key procedural points include purification of naive CD8⁺ T cells, precise gradient control of anti-CD3 coating concentration, and strict normalization of cell number per well. To ensure data comparability across experiments, we also recommend using consistent instrument settings, compensation matrices, and gating strategies, and applying the same antibody lots and fixable viability dye batches where feasible. The reliability of these critical steps directly determines whether experimental results can accurately reflect the effects of varying TCR stimulation intensities on T cell phenotypic dynamics.

In this protocol, we employed plate-bound antibody stimulation, in which anti-CD3 antibodies are directly coated onto the culture plate surface to crosslink the TCR-CD3 complex. This approach offers cost-effectiveness, uniform antibody distribution, minimal inter-well variability, and excellent reproducibility. Notably, the titration of coating antibody concentrations enables precise graded modulation of stimulation intensity, which is a feature that distinguishes this system from bead-conjugated stimulation, where ligand density is typically fixed at a saturating level, limiting fine-tuned signal modulation¹⁴. However, plate-bound stimulation delivers supraphysiological and sustained TCR crosslinking that fundamentally differs from the transient, affinity-dependent engagement induced by peptide-MHC complexes on antigen-presenting cells. While more physiologically relevant, pMHC-based stimulation is technically demanding, requires specialized expertise for multimer production, and incurs substantially higher costs. For cell isolation, we employed negative immunomagnetic selection, which is simple, rapid, and cost-effective. Although it provides lower purity compared with FACS, it avoids the mechanical stress and fluorescent labeling that may compromise the physiological state of sorted cells^20,21.

Key issues that may arise during protocol execution include low sorting purity, weak proliferation, CTV-induced cytotoxicity, poor post-culture viability, and inconsistent staining. For low purity, ensure sufficient mixing of cells with the biotinylated antibody cocktail and streptavidin-coated beads, extend the magnetic separation time if necessary, and optimize the bead-to-antibody ratio for each new reagent lot. If minimal proliferation is observed even at the highest anti-CD3 concentration, verify the viability and purity (especially CD44^hi contamination) of isolated naive cells, confirm the anti-CD3 coating procedure (seal plates with sealing film during overnight incubation at 4 °C and ensure uniform antibody coverage), and validate the IL-2 concentration, as this cytokine is essential for supporting T cell expansion. For CTV-induced cytotoxicity, reduce the staining concentration while maintaining clear resolution of division peaks, and ensure the quenching step is performed strictly on ice for the full 5 min to remove residual free dye^22,23. For poor viability after culture, control cell density, ensure sufficient medium volume, and maintain sterile technique. If inconsistent staining or high flow cytometric variability is observed, prepare antibody cocktails as master mixes, protect all stained samples from light, and use the same antibody lots and fixable viability dye batches across replicates.

Nevertheless, this protocol has certain limitations. As an in vitro system, it cannot recapitulate the complex tissue microenvironment, cellular interactions, or cytokine milieu of secondary lymphoid organs. The culture window is limited to 48 h, restricting analysis to early activation events and precluding assessment of late-stage differentiation, exhaustion, or memory formation. Moreover, the protocol relies heavily on flow cytometric readouts, which demand rigorous standardization of instrument settings, compensation matrices, and gating strategies across independent experiments. Furthermore, the findings obtained with this anti-CD3/CD28-based system are derived solely from CD8⁺ T cells and may not be directly generalizable to CD4⁺ T cells, whose activation thresholds, co-stimulatory requirements, and downstream differentiation trajectories are known to differ^12,24,25. Additionally, the basal activation state of T cells can vary between individual mouse colonies due to differences in commensal microbiota, which constitutes a potential source of inter-laboratory variability that should be considered when interpreting results across studies.

The graded stimulation system described here enables researchers to tailor TCR signal strength to specific experimental objectives based on the distinct response patterns observed across different readouts. This data reveal that the choice of anti-CD3 concentration and harvest time should be guided by the biological question at hand. For immediate early activation markers, CD69 upregulation is detectable as early as 6–12 h across all concentrations but exhibits distinct kinetics: high concentrations (5–10 µg/mL) induce a rapid peak followed by decline, whereas low concentrations (0.5–1 µg/mL) produce a gradual, sustained increase. For proliferation assays, concentrations below 1 µg/mL are suboptimal and may serve as internal negative controls, while 5–10 µg/mL reliably induce robust clonal expansion detectable by CTV dilution at 48 h. The expression of the co-inhibitory receptor PD-1 and the co-stimulatory molecule ICOS requires high-intensity stimulation (5–10 µg/mL) and peaks at 36 h, indicating that studies investigating these molecules should avoid earlier or lower-dose conditions. For effector function readouts (TNF-α and Granzyme B), the relationship between stimulation strength and cytokine production is time-dependent and follows a distinctive biphasic pattern. At 24 h, effector molecule production scales positively with anti-CD3 concentration, with higher doses driving greater cytokine output. By 48 h, however, while the absolute levels of cytokine-producing cells are substantially elevated across all conditions compared to 24 h, the dose-response relationship reverses: lower stimulation concentrations (0.5–1 µg/mL) yield relatively higher frequencies of TNF-α⁺ Granzyme B⁺ cells than high doses (5–10 µg/mL). This inversion suggests that sustained strong TCR signaling, despite promoting initial effector differentiation, may impose a relative constraint on the maintenance of cytokine production capacity per cell, potentially reflecting the onset of activation-induced exhaustion or hyporesponsiveness.

Collectively, these empirical observations position our protocol as a reference framework that allows users to preselect stimulation conditions based on specific downstream endpoints, thereby improving experimental design efficiency and data interpretability. Beyond basic mechanistic studies, this system can also inform optimization of T cell expansion protocols for adoptive immunotherapy, where precise regulation of TCR signal strength may help direct differentiation toward favorable effector or memory phenotypes^26,27,28.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL conical tube	Labgic	CD-002-15A	Sterile 15 mL conical centrifuge tube
4% paraformaldehyde	Beyotime	P0099	4% aqueous fixative for preserving cells before staining
60 mm dish	Labgic	12211	60 mm cell-culture dish
70 µm cell strainer	Labgic	BS-70-CS	70 µm nylon-mesh strainer for preparing single-cell suspensions
96-well round-bottom plate	Beyotime	FPT016	96-well round-bottom plate for cell culture and staining
ACK lysis buffer	Beyotime	C3702	Ammonium-chloride-based red blood cell lysis buffer
anti-mouse CD25	Biolegend	101910	APC-conjugated anti-mouse CD25 antibody, clone 3C7, for flow cytometry
anti-mouse CD28	Bio X Cell	BE0015-1	Purified anti-mouse CD28 antibody, clone 37.51, for T-cell costimulation
anti-mouse CD3	Bio X Cell	BP0001-1	Purified anti-mouse CD3ε antibody, clone 145-2C11, for T-cell activation
anti-mouse CD69	BD Biosciences	553237	PE-conjugated anti-mouse CD69 antibody for activation-marker staining
anti-mouse CD8	Invitrogen	17-0081-82	APC-conjugated anti-mouse CD8a antibody, clone 53-6.7, for flow cytometry
anti-mouse GZMB	Biolegend	372212	PerCP/Cyanine5.5-conjugated anti-human/mouse granzyme B antibody, clone QA16A02, for intracellular flow cytometry
anti-mouse ICOS	BD Biosciences	565886	BV421-conjugated anti-mouse ICOS (CD278) antibody for flow cytometry
anti-mouse PD-1	Biolegend	135220	BV605-conjugated anti-mouse PD-1 (CD279) antibody, clone 29F.1A12, for flow cytometry
anti-mouse TNF-α	Invitrogen	46-7321-82	PerCP-eFluor 710-conjugated anti-mouse TNF-α antibody, clone MP6-XT22, for intracellular flow cytometry
anti-mouse/human CD44	Biolegend	103030	PE/Cyanine7-conjugated anti-mouse/human CD44 antibody, clone IM7, for flow cytometry
biotin anti-mouse CD11b	Biolegend	101204	Biotin-conjugated anti-mouse CD11b antibody for lineage depletion
biotin anti-mouse CD11c	Biolegend	117304	Biotin-conjugated anti-mouse CD11c antibody for lineage depletion
biotin anti-mouse CD16/32	Biolegend	156604	Biotin-conjugated anti-mouse CD16/32 antibody for lineage depletion
biotin anti-mouse CD19	Biolegend	115504	Biotin-conjugated anti-mouse CD19 antibody for lineage depletion
biotin anti-mouse CD4	Biolegend	100508	Biotin-conjugated anti-mouse CD4 antibody for lineage depletion
biotin anti-mouse CD44	Biolegend	103004	Biotin-conjugated anti-mouse CD44 antibody for lineage depletion
biotin anti-mouse CD45R	Biolegend	103204	Biotin-conjugated anti-mouse CD45R/B220 antibody for lineage depletion
biotin anti-mouse CD49b	Invitrogen	13-5971-85	Biotin-conjugated anti-mouse CD49b antibody for lineage depletion
biotin anti-mouse F4/80	Biolegend	123106	Biotin-conjugated anti-mouse F4/80 antibody for lineage depletion
biotin anti-mouse Ly6G	Biolegend	127604	Biotin-conjugated anti-mouse Ly6G antibody for lineage depletion
biotin anti-mouse NK1.1	Biolegend	108704	Biotin-conjugated anti-mouse NK1.1 antibody for lineage depletion
biotin anti-mouse TER119	Biolegend	116204	Biotin-conjugated anti-mouse TER-119 antibody for lineage depletion
C57BL/6 mice	Sibeifu (Beijing) Biotechnology Co., Ltd.	N/A	6–8-week-old male mice used for the experiments
Carbon dioxide (CO2) incubator	Thermo Fisher	3120	Humidified CO2 incubator for mammalian cell culture
CellTrace Violet dye	Invitrogen	C34557	Violet fluorescent dye for monitoring cell proliferation
DMSO	Sigma	D2650	Dimethyl sulfoxide; solvent for preparing reagent stock solutions
Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488	Invitrogen	A-21206	Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H+L) secondary antibody
D-PBS	VivaCell Biosciences	C3590-0500	Dulbecco’s phosphate-buffered saline for washing and reagent preparation
Fetal bovine serum (FBS)	Gibco	C11995500BT	Serum supplement for mammalian cell-culture medium
Fixable Viability Dye	Invitrogen	65-0865-14	Fixable viability dye for excluding dead cells during flow cytometry
Fixation and Permeabilization Solution	BD Biosciences	51-2090KZ	Fixation and permeabilization reagent for intracellular staining
Flat-bottom 96-well plate	Labgic	11510	96-well flat-bottom plate for cell culture and stimulation assays
Flow cytometer	BD Biosciences	LSRFortessa-3 laser	Three-laser flow cytometer for multicolor fluorescence acquisition
Golgi Plug	BD Biosciences	51-2301KZ	Brefeldin A-based protein-transport inhibitor for intracellular cytokine staining
Golgi Stop	BD Biosciences	51-2092KZ	Monensin-based protein-transport inhibitor for intracellular cytokine staining
Ionomycin calcium salt	Sigma	I3909-1ML	Calcium ionophore used with PMA for cell stimulation
Perm/Wash Buffer	BD Biosciences	51-20911KZ	Permeabilization and wash buffer for intracellular staining
Phosflow Lyse/Fix Buffer	BD Biosciences	558049	5× buffer for simultaneous red blood cell lysis and leukocyte fixation
Phosflow Perm/Wash Buffer I	BD Biosciences	557885	Methanol-based permeabilization buffer for intracellular phosphoprotein staining
Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) Rabbit Monoclonal Antibody	Cell Signaling	4858S	Rabbit monoclonal antibody to phospho-S6 ribosomal protein (Ser235/236), clone D57.2.2E
PMA	Sigma	P1585-1MG	Phorbol 12-myristate 13-acetate; activator used for ex vivo cell stimulation
Recombinant Mouse Interleukin-2	Gibco	200-02-50UG	Recombinant mouse interleukin-2 for T-cell culture and expansion
RPMI 1640	Life-Lab	AC01L064	Roswell Park Memorial Institute 1640 medium for mammalian cell culture
Streptavidin Magnetic Beads	BEAVER	22307	Streptavidin-coated magnetic beads for biotin-antibody-based cell depletion
Flow cytometry staining buffer	Not specified	Not specified	Flow cytometry staining buffer; supplier or preparation formula was not provided
Parafilm M laboratory film	Amcor	PM996	Flexible self-sealing laboratory film for sealing plates, tubes, and culture vessels
Lyse/Fix Buffer	BD Biosciences	558049	5× buffer for simultaneous red blood cell lysis and leukocyte fixation
FlowJo	BD Biosciences	v10.8.1	Software, version 10.8.1, for flow cytometry data analysis