T Cell Status

  Comprehensive Analysis of T Cell Status with the NovoCyte Advanteon

T cells play a vital role in controlling infection and cancer, but their activity must be tightly regulated to avoid autoimmunity. A 16-color immunophenotyping panel developed for the NovoCyte Advanteon flow cytometer enables in-depth analysis of T cell differentiation, activation, exhaustion, and functional capacity within a single experiment. 

Key Findings

DT cell differentiation dynamics: Following stimulation, naïve T cells rapidly transitioned into effector and memory subsets, with central memory T cells (TCM) dominating after activation and shifting toward effector phenotypes after restimulation.

Activation markers: CD25 was strongly upregulated, while CD127 was downregulated, consistent with highly activated T cells.

Exhaustion markers: Inhibitory receptors PD-1, LAG-3, and TIM-3 increased after sustained stimulation, reflecting early T cell exhaustion. Interestingly, their expression decreased again after restimulation, coinciding with a shift toward effector status.

Functional output: Activated CD4+ and CD8+ T cells produced TNFα and IFNγ, with most also expressing CD107α, indicating strong cytolytic potential, while IL-2 was minimally detected.



Figure 1. Change of expression level of inhibitory and activation receptors upon stimulation. PBMCs isolated from donor blood were stimulated with 5 µg/mL anti-CD3 antibody, 2 µg/mL anti-CD28 antibody, and 100 ng/mL IL-2. Stimulated PBMCs were stained with the 16-color immunophenotyping panel (Table 1) on days 0, 2, and 4. Cells were restimulated on day 4 and analyzed 24 hours later (day 5). Inhibitory receptors PD-1, LAG-3, and TIM-3 and activation markers CD25 and CD127 were analyzed over time.

Conclusions

The NovoCyte Advanteon’s high-parameter detection capabilities make it a powerful platform for tracking T cell state transitions, from activation to exhaustion. This comprehensive profiling approach provides valuable insight for research in chronic infection, cancer immunology, and checkpoint blockade therapies.


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