Protocol for in vitro isolation, induction, expansion, and determination of human natural regulatory T cells and induced regulatory T cells

1 Hepatobiliary Center of the First Affiliated Hospital, Nanjing Medical University, Research Unit of Liver Transplantation and Transplant Immunology, Chinese Academy of Medical Sciences, Nanjing, Jiangsu 210029, China

Qing Shao

1 Hepatobiliary Center of the First Affiliated Hospital, Nanjing Medical University, Research Unit of Liver Transplantation and Transplant Immunology, Chinese Academy of Medical Sciences, Nanjing, Jiangsu 210029, China

Jinren Zhou

1 Hepatobiliary Center of the First Affiliated Hospital, Nanjing Medical University, Research Unit of Liver Transplantation and Transplant Immunology, Chinese Academy of Medical Sciences, Nanjing, Jiangsu 210029, China

Qiuyang Chen

1 Hepatobiliary Center of the First Affiliated Hospital, Nanjing Medical University, Research Unit of Liver Transplantation and Transplant Immunology, Chinese Academy of Medical Sciences, Nanjing, Jiangsu 210029, China

Ling Lu

1 Hepatobiliary Center of the First Affiliated Hospital, Nanjing Medical University, Research Unit of Liver Transplantation and Transplant Immunology, Chinese Academy of Medical Sciences, Nanjing, Jiangsu 210029, China

1 Hepatobiliary Center of the First Affiliated Hospital, Nanjing Medical University, Research Unit of Liver Transplantation and Transplant Immunology, Chinese Academy of Medical Sciences, Nanjing, Jiangsu 210029, China

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Associated Data

This study did not generate datasets/codes.

Summary

Regulatory T cells (Tregs) can inhibit the occurrence of autoimmune diseases and increase the activation threshold of the immune response. Here, we present schemes for the isolation and culture of natural human regulatory T cells (nTregs) and in vitro-induced Tregs (iTregs). Appropriate concentrations of TGF-β, IL-2, retinoic acid (atRA), and rapamycin were used to promote proliferation to meet sample needs in basic research, especially in technologies such as sequencing.

For complete details on the use and execution of this protocol, please refer to Lu et al. (2014a) and Gu et al. (2022).

Graphical abstract

Highlights

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Regulatory T cells (Tregs) can inhibit the occurrence of autoimmune diseases and increase the activation threshold of the immune response. Here, we present schemes for the isolation and culture of natural human regulatory T cells (nTregs) and in vitro-induced Tregs (iTregs). Appropriate concentrations of TGF-β, IL-2, retinoic acid (atRA), and rapamycin were used to promote proliferation to meet sample needs in basic research, especially in technologies such as sequencing.

Before you begin

Tregs play important roles in autoreactive lymphocyte suppression and regulation of immune homeostasis. This protocol describes a procedure to extract nTreg and naive CD4 + T cells from the peripheral blood of volunteers and induce their expansion.

Due to the low content of nTreg in peripheral blood, we usually extract 100 mL peripheral blood mononuclear lymphocyte cells from the peripheral blood of a single volunteer and then further extract nTreg. To consider the experimental cost, we will extract 100 mL of peripheral blood from volunteers and extract naïve CD4 + T cells (Lu et al., 2010).

Selecting the appropriate culture environment and culture system, and adding the appropriate concentration of cytokines, such as IL-10, TGF-β, atRA, and rapamycin (Lu et al., 2014b), are essential for inducing and expanding Tregs (Lu et al., 2014a). Therefore, before starting, the reagents and equipment listed in the following and attached key resources table should be prepared.

All research work can only be carried out after obtaining the approval of the Institutional Review Board (IRB).

Institutional permissions

Peripheral blood mononuclear lymphocytes from normal volunteers were obtained from the First Affiliated Hospital of Nanjing Medical University under protocols approved by the ethics committee of the First Affiliated Hospital of Nanjing Medical University (2021-SRFA-346).

Preparation of Tregs isolation and culture

Contact volunteers in advance with precautions and signed informed consent forms according to local ethics committee requirements.

Dispense the following required reagents into the corresponding solutions and store them in aliquots.

Note: Transfer of volunteer blood to the laboratory requires a transfer box and ice packs.

Key resources table

Step-by-step method details

Isolation of natural human regulatory T cells (nTregs)

Timing: 3 h for extracting human peripheral blood mononuclear lymphocytes, 3 h for separating nTregs

In this step, human peripheral blood mononuclear lymphocytes were first extracted by MCS®+ 9000 Mobile Platelet Collection System, and then human nTregs were purified by magnetically activated cell sorting (MACS). Alternatively, you can get commercially available buffy coats and skip step 1.

Use MCS®+ 9000 Mobile Platelet Collection System to extract 100 mL PBMCs from the peripheral blood of volunteers.

Place the same volume of lymphoprep ™ below the mixing solution. Indeed the PBS: PBMC is layered ON TOP of the Lymphoprep solution and not vice versa.

CRITICAL: Due to solution density issues, take care of the Lymphoprep™. Make sure it is not mixed with blood and PBS before centrifugation.

Take the middle white film layer after centrifugation (393 g, 23 min, acceleration 0, acceleration 0, 4°C) ( Figure 1 A).

Isolation of natural human regulatory T cells (nTregs) (A) Human peripheral blood mononuclear lymphocytes (PBMCs) were sorted from samples extracted by MCS®+ 9000 Mobile Platelet Collection System. (B) The number of cells obtained by magnetic column sorting at each step. On the first negative selection (NS) step, CD127 - cells were sorted from PBMCs. On the second negative selection step, CD4 + CD127 - cells were got. On the third positive selection (PS) step, CD4 + CD25 + CD127 - nTregs were finally received. (C) The purity of CD4 + CD127 - cells were identified after NS selection, and the purity of CD4 + CD25 + CD127 - nTregs were identified after PS selection on day 0. (D) State of nTregs after selection on day 0 under the microscope (10×).

Wash by adding one volume of PBS (167 g, 5 min, acceleration 9, deceleration 9, 4°C), and collect the cell pellet. (e.g., 1 mL leukocyte suspension with 1 mL PBS).

Add the same volume of PBS as the supernatant, and repeat step.1.d 3–5 times until the liquid was clear.

Note: All of the work with Lymphoprep needs to be at 25°C. Temperature fluctuations will result in poor separation. And all subsequent work (after PBMC isolation) is performed ‘ON ICE’.