Proteins for Immune Cell Culture

CD3⁺ T cells typically make up 70-85% of the lymphocyte population in healthy human adults and 45-70% of peripheral blood mononuclear cells (PBMCs) in healthy individuals. Following PBMC isolation from human blood by Ficoll Hypaque density centrifugation, CD3⁺ T cells can be separated using a magnetic bead cell enrichment kit such as R&D Systems MagCellect^TM Human CD3⁺ T Cell Isolation Kit. Using this negative selection kit, the typical recovery of CD3⁺ T cells ranges from 45-70% and the purity of recovered CD3⁺ T cells is typically greater than 80%.

As the CD3⁺ T cell population includes both CD8⁺ cytotoxic T cells and CD4⁺ T cells, R&D Systems MagCellect^TM Human CD8⁺ T Cell Isolation Kit, MagCellect Human Naïve CD4⁺ T Cell Isolation Kit, MagCellect Human CD4⁺ T Cell Isolation Kit, MagCellect Human Memory CD4+ T Cell Isolation Kit, or MagCellect Human CD4+CD25+ Regulatory T Cell Isolation Kit could also be used to isolate more specific T cell types of interest. Alternatively, T cells can be expanded from PBMCs using R&D Systems ExCellerate^TM Human T Cell Expansion Media, Xeno-Free. Using this protocol, T cells are incubated with CD3/CD28-activating beads or plate-bound antibodies and then cultured in ExCellerate media supplemented with IL-2 for 9 days.

T cells can also be generated from pluripotent stem cells. This involves the co-culture of iPSCs from healthy donors with murine bone marrow stromal cells to generate CD34⁺ hematopoietic progenitor cells. The hematopoietic progenitor cells are then co-cultured with DLL-overexpressing OP9 feeder cells in the presence of SCF, Flt-3 Ligand, IL-7, and IL-15 or IL-21 to promote T cell differentiation.¹ T cells can also be generated from iPSCs derived from peripheral blood T cells in feeder-free culture conditions.² In this protocol, embryoid bodies are generated from single cell-dissociated iPSCs and induced to differentiate into mesoderm and hematopoietic progenitor cells (HPCs) under culture conditions that include BMP-4, FGF-basic, VEGF, and the GSK-3 inhibitor, CHIR99021. HPCs are subsequently differentiated into T cells under feeder-free conditions using immobilized DLL4 and retronectin, in the presence of SCF, TPO, and Flt-3 Ligand, and IL-7.²

Following T cell isolation or expansion, naïve CD4⁺ T helper cells can be differentiated into different T helper cell subsets based primarily upon the addition of specific cytokines to the cell culture media. The different CD4⁺ T helper cell subsets that have been identified include T helper type 1 (Th1), Th2, Th9, Th17, and Th22 cells, along with follicular helper T (Tfh) cells, and regulatory T cells. The table below outlines the cytokines that are required for the differentiation of different Th cell subsets.

The different CD3+ T cell subsets are typically identified by flow cytometry as CD3⁺CD8⁺ T cells, CD3⁺CD4⁺ T cells, or CD3⁺CD4⁺CD8⁺ double positive T cells, with different CD4⁺ T helper cell subsets being distinguished using additional subset-specific cell surface and intracellular markers. Naïve T cells are characterized as CD45RA⁺, CD45RO^-, CD95^-, and CCR7⁺ cells, while stem cell memory T cells (T_SCM) are characterized as CD45RA⁺, CD45RO^-, CD95⁺, and CCR7⁺ cells. In contrast, effector memory T cells (T_EM) are characterized as CD45RO⁺, CD45RA^-, CD62L^-, and CCR7^-, and central memory T cells (T_CM) are characterized as CD45RO⁺, CD45RA^-, CD62L⁺, and CCR7⁺ cells.

References

1. Zhou, Y. et al. (2022) Engineering induced pluripotent stem cells for cancer immunotherapy. Cancers 14:2266.

2. Iriguchi, S. et al. (2021) A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy. Nat. Commun. 12: 430.

Natural killer cells are lymphocytes belonging to the innate immune system that account for 5-20% of the lymphocyte population in humans and up to 15% of peripheral blood mononuclear cells (PBMCs).  Following isolation of PBMCs from human blood by Ficoll Hypaque density centrifugation, natural killer cells can be separated using a magnetic bead cell enrichment kit such as R&D Systems MagCellect^TM Human NK Cell Isolation Kit. Using this negative selection kit, the resulting cell preparation is highly enriched for natural killer cells with the typical purity of the recovered NK cells ranging from 80-90%. Alternatively, natural killer cells can be expanded from isolated PBMCs or CD3⁺-depleted PBMCs using R&D Systems ExCellerate™ Human NK Cell Expansion Media, Animal Component-Free. NK cells are expanded by incubating PBMCs with NK cell activating beads or plate-bound antibody and then culturing the cells in the ExCellerate media supplemented with IL-2, IL-12, IL-18, and IL-21.

Natural killer cells can also be generated from hematopoietic stem cells (HSCs) or from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) after induction of hematopoietic progenitor cells (HPCs). In this type of protocol, iPSCs are differentiated into CD34⁺CD45⁺ hematopoietic progenitor cells, which are then placed on murine feeder cells in medium supplemented with IL-3, IL-7, IL-15, SCF, IL-2 and Flt-3 Ligand to promote NK cell differentiation.^{1, 2} Alternatively, feeder-free hESCs/hiPSCs can be used directly to form embryoid bodies in media supplemented with SCF, VEGF, BMP-4 and ROCK inhibitor followed by NK cell differentiation in media supplemented with IL-3, IL-7, IL-15, SCF, and Flt-3 Ligand.³

Human NK cells are phenotypically characterized as cells expressing high levels of the cell surface marker, CD56/NCAM-1 and lacking expression of CD3. Two different NK cell subsets have been identified in humans. The most abundant NK cell subset found in human blood has been characterized as CD3^-CD56^dimCD16⁺ and is highly cytotoxic, while a second subset that is CD3^-CD56^brightCD16^- has only weak cytotoxic potential and is primarily found in the lymph nodes.

References

1. Euchner, J. et al. (2021) Natural killer cells generated from human induced pluripotent stem cells mature to CD56^brightCD16⁺NKp80^+/- in vitro and express KIR2DL2/DL3 and KIR3DL1. Front. Immunol. 12:640672.

2. Knorr, D.A. et al. (2013) Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy. Stem Cells Transl. Med. 2:274.

3. Zhu, H. and D.S. Kaufman (2019) An improved method to produce clinical-scale natural killer cells from human pluripotent stem cells. Methods Mol. Biol. 2048:107.

CD14⁺ monocytes are present in large numbers in the circulation and the periphery, typically accounting for 10-20% of peripheral blood mononuclear cells (PBMCs) in humans. Following isolation of PBMCs from human blood by Ficoll Hypaque density centrifugation, CD14⁺ monocytes can be separated using a magnetic bead cell enrichment kit such as R&D Systems MagCellect™ Human CD14⁺ Cell Isolation Kit. Using this positive selection kit, the resulting cell population is enriched for CD14⁺ cells with a typical recovery range of 45-75% and typical purity of the recovered cells ranging from 90-97%.

In humans, three subsets of monocytes have been characterized based on the differential expression of the cell surface markers, CD14 and CD16. Classical monocytes are the predominant subset found in human blood and have the phenotype CD14⁺⁺ CD16^-.  The other two smaller monocyte subsets found in humans are the intermediate CD14⁺⁺CD16⁺ monocyte subset and the non-classical CD14⁺CD16⁺⁺ monocyte subset. Together, these two subsets account for less than 15% of the total monocyte population in human blood. 

Once CD14⁺ monocytes are isolated, they can be differentiated into M1 and M2 macrophages using GM-CSF or M-CSF, respectively. Classical M1 macrophage activation is driven by microbial products and pro-inflammatory cytokines such as IFN-gamma and/or lipopolysaccharide (LPS), or TNF-alpha. M1 macrophages typically display a CD14⁺CD80⁺CD163^dimCD206⁺ phenotype and secrete pro-inflammatory factors such as TNF-alpha, IL-1 beta, and IL-12. Alternative M2 macrophage activation is driven by IL-4 and IL-13 and results in the generation of macrophages with an anti-inflammatory phenotype. M2 macrophages typically display a CD14⁺CD80^-CD163⁺CD206⁺ phenotype and secrete anti-inflammatory cytokines such as IL-10 and TGF-beta.

Recently, researchers have also developed protocols to generate macrophages from embryonic or induced pluripotent stem cells in the presence of different combinations of cytokines.¹ Macrophages generated in this way have significant advantages in terms of standardizing the source and scalability, along with being able to further manipulate the cells for the development of macrophage-based cell therapies. Further research is necessary, however, to improve reprogramming methods and to determine the safety of macrophages generated from induced or embryonic stem cells in terms of biodistribution, persistence, and tumorigenicity.¹

References

1. Lyadova, I. and A. Vasiliev (2022) Macrophages derived from pluripotent stem cells: prospective applications and research gaps. Cell Biosci. 12:96.

Monocyte-derived dendritic cells (MoDCs) are a subset of dendritic cells that are widely used in immunology research. Similar to M1 and M2 macrophages, MoDCs are also generated from CD14⁺ monocytes. Following enrichment of CD14⁺ monocytes with R&D Systems MagCellect™ Human CD14⁺ Cell Isolation Kit, monocytes are differentiated into immature MoDCs by culturing the cells with GM-CSF and IL-4. Immature dendritic cells are then activated with pro-inflammatory mediators, such as TNF-alpha or lipopolysaccharide (LPS), to generate mature dendritic cells. Immature dendritic cells typically express low levels of B7-1, B7-2, and MHC class II and they are negative for the cell surface marker, CD83. In contrast, mature dendritic express high levels of B7-1, B7-2, MHC class II, and CD83.

Methods of generating dendritic cells from human embryonic stem cells (hESCs) have also been developed to increase yields, consistency, and expandability compared to MoDCs generated from PBMC-derived CD14⁺ monocytes.¹ These protocols differentiate hESCs into DCs in culture medium supplemented with BMP-4, VEGF, SCF, GM-CSF, and IL-4.¹ Maturation of differentiated immature DCs is achieved by culturing the ESC-derived immature DCs with TNF-alpha, IFN-gamma, PGE₂, and IL-1 beta.

References

1. Silk, K.M. et al. (2011) Differentiation of dendritic cells from human embryonic stem cells.  Methods Mol. Biol. 767:449.

B cells represent around 5-20% of the lymphocyte population in humans and up to 15% of peripheral blood mononuclear cells (PBMCs). Following isolation of PBMCs from human blood by Ficoll Hypaque density centrifugation, B cells can be separated using a magnetic bead cell enrichment kit such as R&D Systems MagCellect^TM Human B Cell Isolation Kit. Using this negative selection kit, the typical recovery ranges from 45-65% and typical purity of the recovered B cells is greater than 80%. B cells can then further be expanded using R&D Systems ExCellerate™ Human B Cell Media, Xeno-Free, supplemented with IL-4 and an anti-CD40 antibody.

There are multiple subsets of B cells in both humans and mice, including naïve B cells, transitional B cells, memory B cells, plasma cells, and regulatory B cells, which can be distinguished based on the expression of different markers. The cell surface marker, CD19 is the most common marker used to identify B cells, as it is stably expressed throughout B cell differentiation until the plasmablast stage. In humans, naïve B cells are typically CD19⁺CD27^-IgD⁺ cells, while transitional B cells are characterized as CD19⁺CD10^+/-CD24^+/- CD27^-IgD⁺. Human memory B cells and plasma cells also express CD19, and in addition, memory B cells are characterized as having high levels of CD27 and low levels of CD23/Fc epsilon RII, while plasma cells are typically characterized as cells expressing high levels of CD38 and CD138/Syndecan-1 and being IgD negative. In contrast, regulatory B cells are a unique subset of immunosuppressive B cells. These cells have been found to express high levels of the cell surface markers, CD21 and CD24, along with CD38, and they secrete IL-10 and TGF-beta.

Granulocytes are polymorphonuclear leukocytes with small cytoplasmic granules that release enzymes to defend the host against invading pathogens. Granulocytes include basophils, eosinophils, mast cells and neutrophils. Of these, neutrophils are the most abundant type of blood leukocyte, but studying them is challenging as they have a short half-life and don’t proliferate, making in vitro expansion impossible.¹ As a result, researchers have developed neutrophil cell lines or have generated neutrophils from induced pluripotent stem cells.^1-3 Several labs have published detailed protocols outlining how to generate functional neutrophils from iPSCs. In general, it is accomplished by co-culturing iPSCs with OP9 cells in the presence of hematopoietic cytokines such as SCF, IL-6, and G-CSF, or IL-3 and G-CSF.^2-4

Eosinophils make up approximately 2-5% of the total peripheral blood leukocyte population in humans, with larger numbers being found in some tissues. They are primarily isolated from a mixed granulocytic population by negative selection using anti-CD16-conjugated magnetic beads or by fluorescence-activated cell sorting (FACS).^5-7 FACS sorting has also been used to study human and mouse bone marrow eosinophils following labeling with a series of eosinophil-specific markers, but a downside of this method is that the antibodies used to label the cells can result in cell activation, which limits the half-life of the cells.⁸ In mouse, eosinophils have also been differentiated in vitro from mouse bone marrow progenitor cells cultured in the presence of SCF, Flt-3 ligand, and IL-5, and human eosinophils have been differentiated from progenitor cells from either the umbilical cord or the bone marrow using IL-3 and IL-5.^8-12 

Mast cells undergo terminal differentiation in tissues and are therefore, found in very low numbers in the circulation. As a result, they are very difficult to isolate and instead, they are typically generated by in vitro differentiation of CD34⁺ hematopoietic progenitor cells from peripheral blood or CD133⁺ progenitors from cord blood.¹³ SCF, IL-3, and IL-6 are used to drive mast cell differentiation of these progenitor cells.

In contrast to mast cells, basophils are primarily found in the circulation rather than in tissues. They typically represent less than 1% of circulating leukocytes. Basophils are most commonly isolated from leukocyte concentrates derived from healthy blood by negative selection using immunomagnetic beads.^{14, 15}

Cell surface and intracellular markers used to identify human or mouse basophils, eosinophils, mast cells, and neutrophils can be found using R&D Systems Cell Marker Interactive Resource Tool and clicking on Granulocytes.

References

1. Blanter, M. et al. (2021) Studying neutrophil function in vitro: Cell models and environmental factors. J. Inflamm. Res. 14:141.

2. Morishima, T. et al. (2011) Neutrophil differentiation from human-induced pluripotent stem cells. J. Cell Physiol. 226:1283.

3. Sweeney, C.L. et al. (2014) Generation of functionally mature neutrophils from induced pluripotent stem cells. Methods Mol Biol. 1124:189.

4. Lachmann, N. et al. (2015) Large-scale hematopoietic differentiation of human induced pluripoteint stem cells provides granulocytes or macrophages for cell replacement therapies. Stem Cell Rep. 4:282.

5. Hansel, T.T. et al. (1989) Purification of human blood eosinophils by negative selection using immunomagnetic beadsv. J. Immunol. Methods 122:97.

6. Hansel. T.T. et al. (1991) An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils. J. Immunol. Methods 145:105.

7. Chihara, J. et al. (1995) A comparative study of eosinophil isolation by different procedures of CD16-negative depletion. Allergy 50:11.

8. Wong, T.W. and D.F. Jelinek (2013) Purification of functional eosinophils from human bone marrow. J. Immunol. Methods 387:130.

9. Dyer, K.D. et al. (2008) Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow. J. Immunol. 181:4004.

10. Ten, R.M. et al. (1991) Eosinophil differentiation of human umbilical cord mononuclear cells and prolonged survival of mature eosinophils by murine EL-4 thymoma cell conditioned medium. Cytokine 3:350.

11. Ohashi, H. et al. (1999) Effect of interleukin-3, interleukin-5, and hyaluronic acid on cultured eosinophils derived from human umbilical cord blood mononuclear cells. Int. Arch. Allergy Immunol. 118:44.

12. Clutterbuck, E.J. et al. (1989) Human interleukin-5 (IL-5) regulates the production of eosinophils in human bone marrow cultures: comparison and interaction with IL-1, IL-3, IL-6, and GMCSF. Blood 73:1504.

13. Radinger, M. et al. (2010) Generation, isolation, and maintenance of human mast cells and mast cell lines. Curr. Protoc. Immunol. Ch. 7:Unit 7.37.

14. Gibbs, B.F. et al. (2008) A rapid two-step procedure for the purification of human peripheral blood basophils to near homogeneity. Clin. Exp. Allergy 38:480.

15. Raap, U. et al. (2016) Human basophils are a source of – and are differentially activated by – IL-31. Clin. Exp. Allergy 47:499.