Description

Human T Regulatory Cells

Human T regulatory cells (Treg cells) are characterized as CD4+ lymphocytes cells that can negatively regulate the activity of T cells, B cells, NK cells, and macrophages. Their suppressive function in lymphoid and non-lymphoid tissues help to establish and maintain tolerance to prevent autoimmunity and hyper-activation in inflammatory responses. Numerous studies have demonstrated the role of Treg cells in the development of autoimmune diseases and efforts are underway to target them with therapeutics to treat these diseases.

Recent studies have also shown that Tregs can infiltrate and develop in the tumor environment. Ovarian, renal, and colon cancers are among the human cancers that have been linked to increased Tregs. Not surprisingly, poor prognosis is associated with an increase in the number of these cells. With these emerging findings, researchers are developing therapeutics that specifically target Tregs to eliminate them from the tumor environment so that remaining lymphocytes can become activated and target tumor cells.

Treg cells come in various subtypes and can develop in the thymus or periphery. Those that develop in the thymus are called Natural Tregs (nTregs), while those that develop in the periphery and tumors are termed Induced Tregs (iTregs). Interestingly, studies have demonstrated that iTregs are more potent at regulating function compared to nTregs. In addition, iTregs can be generated in vitro using a combination of cytokines that drive conventional CD4 cells into iTregs. Both nTregs and iTregs express the surface proteins CD4 and CD25, as well as the transcription factor FOXP3. An additional criteria to identify both types of Tregs is the low expression of CD127, the IL-7 receptor.

Finally, Tregs can suppress lymphocyte function through multiple mechanisms. They can secrete immuno-suppressive factors, such as IL-10 and TGF-b, to prevent the activation or down-regulate the activity of lymphocytes. They may also work by being an “IL-2 sink” in which the high expression of the IL-2 receptor on Tregs bind the local concentrated amount of IL-2 to prevent it from binding lymphocytes to drive an inflammatory response. In addition, Tregs may express checkpoint proteins, such as CTLA-4 and PD-L1, to act through cell-cell contact.

Application Summary for Induced T Regulatory (iTreg) Cells

Induced Tregs are a good source of cells to study the function of Tregs in an in vitro setting, in particular co-culture suppression assays. Here, Tregs are co-cultured with lymphocytes and then assayed for lymphocyte proliferation and cytokine release. In the presence of Tregs, lymphocyte function should be down-regulated.

With the current trend towards development of molecules that modulate the immune system to treat cancer and autoimmune diseases, these co-culture assays are also commonly used to test whether a potential therapeutic can inhibit or promote Treg function. Further, because Tregs are difficult to isolate from tissue and a great number of them are required for co-culture assays, frozen induced Tregs are a valuable source to enable the execution of these studies.

For some antibody-based cancer therapeutics being developed, Tregs are being targeted for antibody-dependent cellular cytotoxicity or complement dependent cytotoxicity. Therefore, frozen iTregs are also a good source of target cells for these studies interrogating cell death.

Many questions about how iTregs develop and progress through developmental stages also remain unanswered, and frozen iTregs are a good source of cells to study this development through genetic and transcriptional profiling. DNA or RNA can easily be isolated from these cells and used for downstream applications and analysis.

Induction/Generation of iTregs

Naive CD4 cells isolated from PBMCs underwent a proprietary process under sterile conditions to generate iTregs. To ensure that Tregs were induced, cells are analyzed for the expression of CD4, CD25, and CD127 (Figure 1). Therefore, our frozen iTregs are a reliable and ideal source to obtain plentiful cells for your studies.

Purity and Function

A small aliquot of cells was analyzed for differentiation into Treg cells by flow cytometry analysis. Characterization of iTreg cells, as defined by CD4 and CD25 expression, was > 94% (Figure 1). Our iTreg cells are also verified by low CD127 expression and confirmed as functionally suppressive.

Figure 1. CD4 and CD25 expression after conversion from conventional CD4 cells. Conventional CD4 (left) cells were converted into iTregs (right) using a proprietary method. A small aliquot was taken after induction to evaluate expression of CD4 and CD25 by flow cytometric analysis (right).

Cryopreservation and Storage

Our iTregs were cryopreserved carefully using iQ Biosciences’ cryopreservation protocol that ensures high viability (> 80%) after thawing.

Cells should be stored at < -120°C once they are received, such as within a liquid nitrogen tank (vapor phase).

Quality Control Process

We implement random sampling per lot to test for viability and cell counts to ensure they meet specifications, which are recorded on the Certificate of Analysis that is included with each shipment. Cell counts are obtained using a manual hemocytometer and then cross-referenced with an automated cell counter. Each lot is also characterized for unique cell populations by immunophenotyping, in which the results are also recorded on the Certificate of Analysis.