Impact of PMA Stimulation on CD4 Detection and Solutions

Many researchers have faced a challenge when detecting cells such as Th1/Th2/Th17, where direct CD4 detection shows clear cell subgrouping. However, for cytokine detection, PMA stimulation is necessary before CD4 detection. After stimulation, the CD4-positive and CD4-negative cell populations become less distinguishable.

· What is the cause of this phenomenon?

· What are the potential solutions?

Today, let's delve into these questions:

CD4 Expression in Peripheral Blood Before and After PMA Stimulation

In fact, as early as 1990, a study by Michael Bigby revealed the influence of PMA stimulation on CD4 expression ("Phorbol myristate acetate-induced down-modulation of CD4 is dependent on calmodulin and intracellular calcium." The Journal of Immunology, 1990, 144:3111-3116).

The study demonstrated CD4 expression in six cell types and the changes in CD4 expression after stimulating these cells with PMA concentrations ranging from 5 ng/mL to 500 ng/mL. The results showed that Sub T1, MOLT-3, peripheral blood lymphocytes, mouse thymocytes, and AKR 1G1 cells exhibited significant CD4 internalization following PMA stimulation, except for mouse peripheral blood lymphocytes, where CD4 expression change was minimal. 

Effects of PMA Stimulation on CD4 Expression

To further understand the mechanism of CD4 internalization caused by PMA stimulation, the authors used calcium calmodulin inhibitor W-7 and trifluoperazine. Both substances effectively inhibited PMA-induced CD4 internalization at a concentration of 25 μM, suggesting the involvement of calcium ions in CD4 internalization. 

Effects of W-7 or Trifluoperazine on PMA-Induced CD4 Internalization

Does intracellular calcium ions or extracellular calcium ions play a crucial role? To test this, the authors used calcium ion chelators EGTA and QUIN-2. 

Effects of Intracellular and Extracellular Calcium Ions on PMA-Induced CD4 Internalization

EGTA chelates extracellular calcium ions, while QUIN-2 AM binds to intracellular calcium ions. Results showed that QUIN-2 AM-treated cells, lacking intracellular calcium ions, did not exhibit CD4 internalization after PMA stimulation, indicating the importance of intracellular calcium ions in PMA-induced CD4 internalization.

Based on these experimental results and our experience, we propose several solutions for addressing PMA-induced CD4 internalization:

✦ Choose brighter fluorescent labels or clones with higher conjugation efficiency.

✦ Combine CD4 and CD8 gating to enhance the purity of CD4+ T cells in the gated population.

✦ Consider using the calcium ion chelator QUIN-2 AM to prevent CD4 downregulation, ensuring it doesn't affect other measurements.

Recently, we have introduced new clones SK3 and RM4-5 for Human CD4 and Mouse CD4, respectively, which exhibit reduced internalization effects. For more details, refer to the table below: