Committed to providing comprehensive support for global drug development

Interleukin-2 (IL-2) is an O-glycosylated cytokine characterized by a four-alpha-helix bundle structure, primarily produced by activated T cells, dendritic cells, and B cells. As a potent inducer of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, IL-2 was one of the first immunotherapeutic agents approved for the treatment of metastatic melanoma and renal cell carcinoma. However, due to its short in vivo half-life and severe toxicity at therapeutic doses, IL-2 immunotherapy has not been widely adopted since its initial approval. The IL-2 receptor (IL-2R) consists of three subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). The binding of IL-2 to CD25 induces the proliferation of regulatory T cells (Tregs)—a phenomenon attributed to the preferential expression of CD25 on Tregs. Since the depletion of Tregs has been shown to enhance IL-2-induced anti-tumor immunity, this suggests that Tregs may constitute a major barrier to IL-2-mediated CTL expansion.

IL-2 exerts its stimulatory and regulatory functions by binding to various types of IL-2 receptors, including monomeric, dimeric, and trimeric forms. Certain T cells—such as Tregs—express a high-affinity heterotrimeric receptor composed of the CD25, CD122, and CD132 subunits. Naive CD8+ T cells, CD4+/CD8+ memory T cells, and NK cells express a lower-affinity dimeric receptor consisting of the CD122 and CD132 subunits, which lacks the CD25 subunit. The affinity of the dimeric receptor for IL-2 is merely one-hundredth that of the complete trimeric receptor. When CD25 exists in isolation, it exhibits the lowest affinity and does not transduce intracellular signals; the activation of downstream signaling pathways is strictly dependent on the presence of the CD122 and CD132 subunits, while the primary function of CD25 is to facilitate the binding of IL-2. IL-2 is initially captured by CD25, inducing a conformational change in IL-2 that increases its affinity for CD122 and CD132. This leads to the dimerization of the signaling motifs within the cytoplasmic tails of CD122 and CD132, resulting in the phosphorylation of the Janus kinases JAK1 and JAK3, which subsequently exert kinase activity on key tyrosine residues located within the tail of the CD122 subunit. There are three primary downstream signaling pathways: the JAK-STAT pathway, the Phosphoinositide 3-kinase (PI3K)-AKT pathway, and the Mitogen-activated protein kinase (MAPK) pathway.

The Jurkat E6.1 IL2/IL15 Dual Effector (mCD25/hCD122/mCD132) Reporter Cell Model—effectively simulates the signal transduction process of  IL2  *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Jurkat E6.1 IL2/IL15 Dual Effector (mCD25/hCD122/mCD132) Reporter Cell Model

Figure 2. Dose Response of Recombinant Human IL2 in hIL2/IL15 (mCD25hCD122mCD132) Effector Reporter Cell (C31). Dose Response of Recombinant Human IL15 in hIL2/IL15 (mCD25 hCD122 mCD132) Effector Reporter Cell(C31).

Cell Passage Procedures

1.This cell line grows in suspension.
2.Upon receipt, cells should be thawed immediately or stored in liquid nitrogen until use.
3.Before thawing, pre-warm the water bath and culture medium to 37 °C, and prepare a small amount of dry ice.
4.Remove the cryovial from storage and transport it to the cell culture laboratory on dry ice.
5.Rapidly thaw the cells in a 37 °C water bath. Once the cells are completely thawed, spray the cryovial with 70% ethanol for disinfection and transfer it to a biosafety cabinet.
6.Add 10 mL of pre-warmed culture medium into a 15 mL centrifuge tube. Transfer the contents of the cryovial into the tube and centrifuge at 1000 rpm for 5 minutes.
7.Carefully discard the supernatant. Resuspend the cell pellet in 5 mL of pre-warmed culture medium by gentle pipetting. Immediately perform cell counting and adjust the cell density to 3–6 × 10⁵ cells/mL based on the counting results, then transfer the cells into a culture flask.
8.Count the cells every 1–2 days. When the cell density exceeds 1 × 10⁶ cells/mL, passage the cells promptly or add fresh culture medium. Maintain the cell density between 2 × 10⁵ and 1 × 10⁶ cells/mL.

Suspension Cell Cryopreservation Procedure:

1.Collect 8 × 10⁶ cells, centrifuge, and discard the supernatant.
2.Add 1 mL of cell freezing medium (90% FBS + 10% DMSO) and gently pipette to mix thoroughly. Transfer the suspension into a cryovial.
3.Immediately place the cryovial into a controlled-rate freezing container (Nalgene 5100-0001), fill with isopropanol up to the indicated level, and store at −80 °C.
4.After 24 hours, transfer the cryovial to liquid nitrogen for long-term storage.