Defining Human PBMC T cell activation markers. Part 2: CD71 and CD95

In a previous posting, I discussed the use of T cell activation markers as a strategy for assessing the function of T cells from human peripheral blood mononuclear cells (PBMC). Following T cell receptor (TCR) activation, T cells will express a series of activation markers that include chemokine and cytokine receptors, adhesion molecules, co-stimulatory molecules, and MHC-class II proteins. Understanding what these activation markers are, when they are expressed, and their role in T cell function during normal responses and disease states is important when selecting markers for assessing T cell biology for studies on human PBMC.

In the previous posting, I discussed two immediate early activation markers for assessing the activation status of human PBMC T cells: CD69 and CD40L.  In this article, the second in this series, I will discuss two additional mid-early T cell activation markers that can be assessed by flow cytometry: CD71 and CD95.

CD71 (TFRC, Transferrin Receptor, TfR) is a cell surface iron transport receptor that is upregulated in proliferating cells by 24-48 hours following T cell activation and expression continues to rise and is maintained for several days.  Thus CD71 can be considered a mid-early activation marker as compared with late activation markers that are not appreciably upregulated until even later time points.  CD71 has been shown to associate with the TCRz chain and ZAP70 and may participate in TCR signaling, and is an essential factor for proliferating T cells.

The inability of CD71 to be upregulated following TCR activation may be associated with T cell dysfunction.  As was similarly discussed for CD69, Critchley-Thorne et. al, 2007 showed that PBMC T cells from metastatic melanoma patients had reduced CD71 upregulation compared with healthy controls, and this corresponded with multiple other functional defects in T cells from these patients.  Thus CD71 may be aberrantly expressed by T cells in human disease.

fas signalingCD95 (Fas, APO-1, TNFRSF6) is a member of the TNF-receptor superfamily and is best known for its role in mediating activation-induced cell death in activated T cells following binding to its ligand, CD95L/FasL induced on antigen-presenting cells (APCs).  However, CD95 can also play additional, non-apoptotic roles in the modulation of T cell function.  CD95 ligation has been shown to inhibit TCR signaling and activation of naïve T cells.  However, this negative co-stimulatory effect appears to be dose-dependent, as low doses of CD95 agonists had the opposite effect and strongly promoted activation and proliferation of T cells.  Like CD71, CD95 expression can be detected by 24 hours following T cell activation and continues to increase over the course of several days.

Due to its differential roles in regulation of T cell apoptosis and activation, dysregulated expression of CD95 or its ligand CD95L could be avenues for T cell dysfunction in various human diseases.  Indeed, Strauss et. al, showed that regulation of CD95L expression may play a role in immune evasion during viral infections. CD95L was upregulated in HIV-infected APCs, and led to suppressed T cell activation.  Interferons are known to enhance CD95 expression, and our group (Critchley-Thorne et. al, 2009) has shown reduced upregulation of CD95 in PBMC T cells from breast cancer patients following T cell activation in the presence of interferons, indicating the lack of full T cell activation under these conditions.

Thus both CD71 and CD95 are upregulated in the mid-early phase of T cell activation and dysfunctional expression may be useful measures of T cell dysfunction in various disease states. Thus, these may be useful markers when assessing the phenotype and function of human PBMCs.


Additional Reading:

Comparative analysis of lymphocyte activation marker expression and cytokine secretion profile in stimulated human peripheral blood mononuclear cell cultures: an in vitro model to monitor cellular immune function.  Reddy M, Eirikis E, Davis C, Davis HM, Prabhakar U. J Immunol Methods. 2004 Oct;293(1-2):127-42.

Multiparametric flow cytometric analysis of the kinetics of surface molecule expression after polyclonal activation of human peripheral blood T lymphocytes. Biselli R, Matricardi PM, D’Amelio R, Fattorossi A. Scand J Immunol. 1992 Apr;35(4):439-47.

Surface markers of lymphocyte activation and markers of cell proliferation.  Shipkova M, Wieland E.  Clin Chim Acta. 2012 Sep 8;413(17-18):1338-49.

Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.  Caruso A, Licenziati S, Corulli M, Canaris AD, De Francesco MA, Fiorentini S, Peroni L, Fallacara F, Dima F, Balsari A, Turano A.   Cytometry. 1997 Jan 1;27(1):71-6.

Transferrin receptor induces tyrosine phosphorylation in T cells and is physically associated with the TCR zeta-chain.  Salmerón A, Borroto A, Fresno M, Crumpton MJ, Ley SC, Alarcón B. J Immunol. 1995 Feb 15;154(4):1675-83.

Transferrin synthesis by inducer T lymphocytes.  Lum JB, Infante AJ, Makker DM, Yang F, Bowman BH. J Clin Invest. 1986 Mar;77(3):841-9.

Down-regulation of the interferon signaling pathway in T lymphocytes from patients with metastatic melanoma.  Critchley-Thorne RJ, Yan N, Nacu S, Weber J, Holmes SP, Lee PP. PLoS Med. 2007 May;4(5):e176.

Pro- and anti-apoptotic CD95 signaling in T cells.  Paulsen M, Janssen O. Cell Commun Signal. 2011 Apr 8;9:7.

CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling.  Strauss G, Lindquist JA, Arhel N, Felder E, Karl S, Haas TL, Fulda S, Walczak H, Kirchhoff F, Debatin KM.  J Exp Med 2009, 206:1379-1393.

Impaired interferon signaling is a common immune defect in human cancer.  Critchley-Thorne RJ, Simons DL, Yan N, Miyahira AK, Dirbas FM, Johnson DL, Swetter SM, Carlson RW, Fisher GA, Koong A, Holmes S, Lee PP. Proc Natl Acad Sci U S A. 2009 Jun 2;106(22):9010-5.

*Image courtesy of*

Generation of Dendritic Cells from Peripheral Monocytes

describe the imagePBMCs are not just a source of many different circulating immune cell types, but also a source of potential cells that one can generate in vitro. One excellent and long-standing example of this is the generation of dendritic cells (DCs) from monocytes.  Monocyte derived DCs (mDCs) are an excellent tool for researchers to do immunological assays requiring a source of professional antigen presenting cells (APCs). While circulating B cells are capable of antigen presentation and T cell activation, they do not offer the robust response that DCs do. The generation of mDCs is a relatively simple protocol that anyone can do with just a source of PBMCs, a few important cytokines, and, of course, some media and incubator space. After this protocol, you will have obtained immature mDCs that can then be matured for use as APCs in your assay.

The first step in generating mDCs is to decide how you would like to isolate the monocyte population from your PBMCs, which serve as your precursor cells for DCs. The easiest and cheapest way is to simply plate your PBMCs on a cell culture dish and let the inherent qualities of monocytes go to work. Monocytes are unique amongst other PBMC cells in their tendency to stick to plastic. An incubation period between 1-24 hours will allow your monocytes to adhere to the dish and let you gently wash off any other PBMCs. The alternative to the adherence method for isolating monocytes is to use a magnetic antibody based technology of your choice. Several companies, such as Miltenyi Biotec, Life Technologies, and Stem Cell Technologies, offer excellent kits for this. While the adherence method is cheaper, antibody based kits give you higher monocyte recovery and purity, which may or may not matter depending on your downstream assays.

Once you have your monocytes isolated from your PBMCs, you can begin the 7 day culture to generate mDCs. Monocytes can be plated in a standard cell culture media along with two important cytokines, GM-CSF and IL-4 (50ng/mL and 100ng/mL). GM-CSF will push the monocytes down a DC differentiation pathway. IL-4 will inhibit the monocytes from differentiating into macrophages, thereby insuring they become DCs. Continue the culture for 6-8 days and be sure to refresh your cytokines every other day.

As the monocytes differentiate over the culture period, note their progress by examining them with your tissue culture room microscope. The cells should appear as fairly round and are generally 2-3 times the size of lymphocytes. It is important to note that the mDCs will not appear like the elongated cartoon DCs with long extensions you see in text books. Those DC characteristics are generally only found in tissues and not in vitro.  While you may see some cells that resemble this, those are more likely to be somewhat of a natural stromal layer, made up of cells including macrophages, that the monocyte culture develops to support cell growth. In fact, the immature mDCs will have very few if any, cytoplasmic protrusions.

DC2 resized 600Once the culture period has finished, between 6-8 days, the mDCs can be collected. The exact day is not critical, as long as you remain consistent in the day you pick for your following experiments. To collect the mDCs, gently wash the culture dishes with several streams of media by pipetting up and down. The mDCs, which are currently immature, will be somewhat floating and only loosely adherent. Because of their loose adherence, they require several rounds of gentle pipetting, but do not require cell scraping, EDTA, or trypsin treatment. Note that the culture dishes will still contain some adherent cells. Do not worry about these cells, since these are not the loosely adherent DCs we are interested in.

After completion of these steps, you should have a nice population of immature mDCs, which express CD11c, CD1c, and are CD123-. In my next post, I will cover some tips and tricks for analyzing these cells by flow cytometry. Importantly, I will also cover ways to mature the immature mDCs for use as APCs.

Colt EgelstonColt Egelston is currently a post-doctoral fellow at the Beckman Research Institute of the City of Hope, in Duarte, CA. He received his Ph.D. from Rush University in Chicago and is interested in all things immunology.



Human PBMC T cell immediate early activation markers: What are they and what do they do?

melanoma dividing cellsThere are many strategies for assessing the function of T cells from human peripheral blood mononuclear cells (PBMC).  T cells that have recently been activated through their T cell receptor (TCR) will express a series of activation markers at different time points following activation.   Activation markers include receptors such as chemokine and cytokine receptors, adhesion molecules, co-stimulatory molecules, and MHC-class II proteins.  Some of these molecules have established functions in T cell biology, while the relevance or function of others remains elusive.  Flow cytometry is the method of choice for evaluating various types of surface or intracellular markers that indicate the activation status of T cells.  However, what are these markers, what is their function in T cell biology, what T cell populations will express them, and when can they be assessed are key questions to address when deciding which markers are best for a given assay and question of interest.

In this article, the first of a short series, I will discuss two of the most commonly used immediate early activation markers for assessing the activation status of human PBMC T cells: CD69 and CD40L.

Immediate Early Activation Markers:

CD69 (AIM, Leu23, MLR3) is a signaling membrane glycoprotein involved in inducing T cell proliferation. CD69 is expressed at very low levels on resting CD4+ or CD8+ T cells in PBMC (<5-10%), and is one of the earliest assessable activation markers, being rapidly upregulated on CD4+ or CD8+ T cells within 1 hour of TCR stimulation or other T cell activators such as phorbol esters via a protein kinase C (PKC) dependant pathway.  Expression of CD69 peaks by 16-24 hours and then declines, being barely detectable 72 hours after the stimulus has been withdrawn.

The inability to upregulate CD69 following TCR activation may be associated with T cell dysfunction.  For instance, Critchley-Thorne et. al, showed that PBMC T cells from metastatic melanoma patients with lower responsiveness to interferons had reduced CD69 upregulation compared with healthy controls, and this corresponded with multiple other functional defects in T cells from these patients.  Thus CD69 expression may be a measure of T cell dysfunction in human disease.

CD40L (CD154) is a member of the TNF-receptor superfamily that functions as a co-stimulatory molecule by binding CD40 which is constitutively expressed on antigen presenting cells (APCs).  The CD40L-CD40 ligation results in the activation of multiple downstream pathways including the MAPK (JNK, p38, ERK1/2), NF-ĸB, and STAT3 transcription factors.  CD40L expression is quickly upregulated within 1-2 hours after TCR stimulation via the transcription factors NFAT and AP-1.  CD40L expression peaks near 6 hours after stimulation, and declines by 16-24hrs. CD40L expression however is biphasic, and the addition of anti-CD28 or IL-2 along with TCR stimulation leads to sustained expression for several days (Snyder et. al., 2007).

Expression of CD40L on resting PBMC CD4+ or CD8+ T cells from healthy donors is very low (<1%).  However this percentage has been shown to be significantly increased on up to 17% of CD4+ T cells and 21% of CD8+ T cells in patients with active SLE, and these differences between healthy and SLE patients were also seen following anti-CD3 stimulation of PBMCs (Desai-Mehta, et. al, 1996).  The review below by Daoussis et. al, discusses the role of CD40L expression in several other human diseases.

In summary, CD69 and CD40L are both rapidly induced following T cell activation and both exert important functions in T cell biology. Expressions of these markers have both been shown to be altered in various human diseases.  Understanding the biology of T cell activation markers will allow for the best application of these markers to specific experimental questions and assay types.


Additional Reading:

Multiparametric flow cytometric analysis of the kinetics of surface molecule expression after polyclonal activation of human peripheral blood T lymphocytes. Biselli R, Matricardi PM, D’Amelio R, Fattorossi A. Scand J Immunol. 1992 Apr;35(4):439-47.

Surface markers of lymphocyte activation and markers of cell proliferation.  Shipkova M, Wieland E.  Clin Chim Acta. 2012 Sep 8;413(17-18):1338-49.

Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.  Caruso A, Licenziati S, Corulli M, Canaris AD, De Francesco MA, Fiorentini S, Peroni L, Fallacara F, Dima F, Balsari A, Turano A.   Cytometry. 1997 Jan 1;27(1):71-6.

T cell activation via Leu-23 (CD69).  Testi R, Phillips JH, Lanier LL. J Immunol. 1989 Aug 15;143(4):1123-8.

A whole-blood assay for qualitative and semiquantitative measurements of CD69 surface expression on CD4 and CD8 T lymphocytes using flow cytometry.  Lim LC, Fiordalisi MN, Mantell JL, Schmitz JL, Folds JD. Clin Diagn Lab Immunol. 1998 May;5(3):392-8.

Utility of flow cytometric detection of CD69 expression as a rapid method for determining poly- and oligoclonal lymphocyte activation.  P E Simms and T M Ellis.  Clin Diagn Lab Immunol. 1996 May; 3(3): 301–304.

Down-regulation of the interferon signaling pathway in T lymphocytes from patients with metastatic melanoma.  Critchley-Thorne RJ, Yan N, Nacu S, Weber J, Holmes SP, Lee PP. PLoS Med. 2007 May;4(5):e176.

Direct inhibition of CD40L expression can contribute to the clinical efficacy of daclizumab independently of its effects on cell division and Th1/Th2 cytokine production.  Snyder JT, Shen J, Azmi H, Hou J, Fowler DH, Ragheb JA. Blood. 2007 Jun 15;109(12):5399-406.

Targeting CD40L: a Promising Therapeutic Approach.  D. Daoussis, A.P. Andonopoulos, and S. C. Liossis. Clin Diagn Lab Immunol. 2004 July; 11(4): 635–641.

Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. J. Clin. Investig. 97:2063-2073. Desai-Mehta, A., L. Liangjun, R. Ramsey-Goldman, and S. Datta. 1996.

Photo credit: wellcome images / / CC BY-NC-ND

Weird but common T cell populations in human PBMC

Human T cells are generally analyzed for expression of CD4 and CD8 to classify them as either of these two major classes of effector T cells.  But flow cytometry analysis of PBMCs stained with antibodies targeting CD3, CD4, and CD8 reveals several other populations with varying expression of these three markers.  So what are they?

For the sake of this discussion, I will refer to the largest typical single positive populations of CD3+CD4+ and CD3+CD8+ T cells as CD4high and CD8 high, respectively.

These are some other T cell populations that have been observed in human PBMC:

CD8lowCD4high (1): Populations of CD4 T cells that express CD8.  This population is likely heterogeneous, and compared with the CD4high population, includes a higher proportion of effector memory and terminally differentiated effector CD4 T cells that have re-expressed CD8.  CD8 is expressed as a heterodimer of either α/α, α/β, or β/β, and this population has been noted to be primarily CD8α/α.  Work by Zloza et. al, identified that up to 50% of these cells can be NKT cells, including invariant CD3+6B11+ NKT cells and non-invariant CD3+CD16/56+ NKT cells.  Of note, NKT cells may also be present at low frequencies in CD4+CD8+, CD4CD8, CD4 or CD8 single positive populations.

CD4lowCD8high (2): Populations of CD8+ T cells, of the primarily CD8α/β type that express CD4.   This population can be further subdivided into two groups:  CD4dimCD8high and CD4medCD8high.  Studies have shown that expression of CD4 on these CD8+ T cells is functional and inducible by stimulations such as anti-CD3/CD28.   These cells express markers of activated T cells and exhibit a higher frequency of memory cells (CD45RA) as compared with typical CD8high cells.

CD8low (3): These cells express CD8 at lower levels compared with CD8 high populations, are negative for CD4 expression, and can express higher levels of CD3.  Trautmann et. al, describe the frequency of CD8 low cells as being from 0.2%-7% of CD8 T cells in healthy donors and described these cells as populations of oligoclonal cytotoxic terminally differentiated effector CD8 T cells (CD45RA+CD62L).

WeirdCD3CD4CD8 Tcell Populations resized 600

CD4neg CD8neg CD3high (4):  CD4 and CD8 double negative cells that express high levels of CD3 compared with CD4 high and CD8 high populations.  This fraction has been shown to contain largely the TCRγ/δ T cell subset although γ/δ T cells can express and the CD8α and/or the CD8β chains.

CD4neg CD8neg CD3pos (5): This fraction has been shown to largely contain heterogeneously differentiated TCRα/β T cell subsets including regulatory T cells.  The expression of CD3 on this subset is lower than that of the CD4neg CD8neg CD3high subset containing γ/δ T cells, although γ/δ T cells may be present in this population as well.

An important thing to note is that characterizations of these populations are generalizations and individuals have been shown to have aberrant profiles compared with these.  Other populations have been described such as CD4high CD8high double positive cells which may be primarily effector memory T cells but here I have focused on those populations I see most frequently.   In summary, careful gating and analyses of each of these populations is necessary, as these are not only functionally unique subsets, but each population appears to be heterogeneous and also contain varying percentages of NKT cells.


Further Reading:

CD4(+)CD8(dim) T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV-1 antigens.  Suni MA, Ghanekar SA, Houck DW, Maecker HT, Wormsley SB, Picker LJ, Moss RB, Maino VC. Eur J Immunol. 2001 Aug;31(8):2512-20.

Multiple populations of T lymphocytes are distinguished by the level of CD4 and CD8 coexpression and require individual consideration.  Zloza A. and Al-Harthi, L. Journal of Leukocyte BiologyJ Leukoc Biol. 2006 Jan;79(1):4-6.

Characterization of circulating CD4+ CD8+ lymphocytes in healthy individuals prompted by identification of a blood donor with a markedly elevated level of CD4+ CD8+ lymphocytes.  Prince HE, Golding J, York J. Clin Diagn Lab Immunol. 1994 Sep;1(5):597-605.

Upregulation of CD4 on CD8+ T cells: CD4dimCD8bright T cells constitute an activated phenotype of CD8+ T cells. Sullivan YB, Landay AL, Zack JA, Kitchen SG, Al-Harthi L. Immunology. 2001;103: 270-280.

Human CD8 T cells of the peripheral blood contain a low CD8 expressing cytotoxic/effector subpopulation.  Trautmann A, Rückert B, Schmid-Grendelmeier P, Niederer E, Bröcker EB, Blaser K, Akdis CA. Immunology. 2003 Mar;108(3):305-12.

CD3 bright lymphocyte population reveal gammadelta T cells.  Lambert C, Genin C. Cytometry B Clin Cytom. 2004 Sep;61(1):45-53.

Isolation and characterization of human antigen-specific TCR alpha beta+ CD4(-)CD8- double-negative regulatory T cells.  Fischer K, Voelkl S, Heymann J, Przybylski GK, Mondal K, Laumer M, Kunz-Schughart L, Schmidt CA, Andreesen R, Mackensen A. Blood. 2005 Apr 1;105(7):2828-35.

Distinct CD4+ CD8+ double-positive T cells in the blood and liver of patients during chronic hepatitis B and C. Nascimbeni M, Pol S, Saunier B. PLoS One. 2011;6(5):e20145.

CD4+ CD8+ double positive (DP) T cells in health and disease.  Parel Y, Chizzolini C. Autoimmun Rev. 2004 Mar;3(3):215-20.

High Throughput Systems for Maximizing Human PBMC Assay Potential

Humans are a heterogeneous population and studies comparing populations of humans require a high number of samples for statistical validity.  In addition, human samples such as PBMC are precious in that they represent the immune state of an individual at a point in time.  Thus, when studies are done to analyze a particular state of the immune response in individuals, such as pre- versus post-vaccination, or along the course of a disease state, once used, the samples can never be replaced.  To make the most of human PBMC samples, in particular when patient samples are being used, it is important to not only carefully optimize assays, but additionally be able to maximize the questions that can be addressed with these samples.

Having recently completed a large study involving human patient PBMCs, I encourage the use of high throughput assays systems that allow for a streamlined experimental approach.  All of these assays involve 96-well plate based methods and commercially available kits.


Basic Equipment for 96-well Plate Assays:

Multichannel Pipettes are necessary for quickly performing all 96-well plate assays.  These come in p1000, p200, p20, and p2 volumes.

96 well plate96-well Plates:  Different types of 96-well plates are available for different assay types.  There are various surface coatings including tissue-culture treated polystyrene for cell cultures, uncoated, and others.  Plates can have various plate bottom geometries and optical characteristics.  For instance there are black plates available for light-sensitive assays.  For protocols involving volumes larger then 250ul, there are deep-well plates that carry a 2ml volume per well.

VPscientific multichannelMultichannel Vacuums: Companies such as V&P Scientific offer a multitude of multichannel vacuum manifolds that fit plates of different depths for removing supernatant from wells via vacuum apparatus.  Often these will be the proper length such that they don’t touch the well bottom and work well with removing buffers from centrifuged PBMC cell cultures, such as during washing steps for flow-cytometry.


PBMC subset Purification:  For magnetic bead based purification of PBMC populations of interest, Stem Cell Technologies offers a 96-well plate EasyPlate™ EasySep™ Magnet that allows separation of up to 1 x 107 cells per well.  Currently only negative or untouched cell isolation methods are supported by this magnetic system due to the larger size of the magnetic beads used in Stem Cell Technologies’ negative isolation kits compared with positive isolation kits.


RNA Isolation:  Qiagen offers two kits for 96-well purification of total RNA from cells.  The RNeasy 96 Kit and RNeasy Plus 96 Kit.  These are 96-well column based platforms which require either a Qiagen vacuum manifold or specialized centrifuge for the protocol.  The RNeasy 96 Kit and RNeasy Plus 96 Kit are similar with the RNeasy Plus 96 Kit utilizing an extra set of steps and columns for elimination of genomic DNA.  The standard RNeasy 96 Kit protocol does however have an optional step for on-column DNAse digestion, however DNAse is not included in the kit.

RNeasy 96 Kit:

RNeasy Plus 96 Kit:


RNA Quantification is much easier if done by 96-well methods than one sample at a time.  Life Technologies’ Quant-iT™ RiboGreen® RNA Assay Kit is extremely sensitive but requires a fluorescence-plate reader.  Thermo Scientific now has a NanoDrop 8000 UV-Vis Spectrophotometer that quantifies nucleic acid concentrations from 96-well plates.


In summary, systematic high-throughput protocols can be developed using 96-well systems such as these and many others.  Thus, numerous PBMC samples can be put through multiple experimental procedures in a streamlined manner, maximizing efficiency and minimizing experimental variation.  In this way, multiple questions can easily be simultaneously addressed in precious PBMC samples.

Markers for Identification of Regulatory T cells in Human PBMC

Forkhead box P3 (FoxP3)+ CD4+ T cells, known as regulatory T cells or TREGs, are a class of negative regulatory T cells that function to suppress immune responses, thereby establishing tolerance, preventing autoimmunity, and allowing tumor escapes from immune surveillance.   TREGs are thought to be generated by two major mechanisms.  Natural TREGs are generated through positive selection in the thymus via differential TCR signaling compared with conventional T cells.  Adaptive or converted TREGs are thought be generated in the periphery by conversion of conventional CD4+ T cells via various mechanisms.

TREGs are a heterogeneous population of T cells that function via cell-contact dependent and independent mechanisms to suppress various immune cell types.  Contact-dependent mechanisms of suppression include expression of negative regulatory receptors such as CTLA4, or killing of associated dendritic cells (DCs) through secretion of perforin and granzyme B.  Contact-independant mechanisms of suppression include TREGs secretion of immune suppressive cytokines including IL-10 and TGFb.   High expression of the IL-2 co-receptor CD25 allows TREGs to act as a sink for IL-2 thereby leading to IL-2 deprivation of conventional T cells and inhibition of proliferation.

TREGs are thus an important class of cells and study of these cell populations in human PBMC requires an understanding of the surface and intracellular markers that can be used for flow cytometry analysis and isolation by Fluorescence-activated cell sorting (FACS) or other methods.

Miyara et. al. identified three functionally unique FoxP3+ populations in  freshly isolated CD4+ T cells from human PBMC.  These three populations could be identified by flow cytometry staining of CD45RA, FoxP3, and CD25.  CD25 and FoxP3 expression were highly correlated in the CD4+ population, and I have consistently seen this in my own analyses of unstimulated human PBMC.  The three populations included CD45RA+FoxP3low cells which were CD25++, CD45RAFoxP3high cells which were CD25+++, and CD45RAFoxP3low cells which were CD25 ++.  When these populations were FACS sorted based on CD45RA and CD25 expression, only CD45RA+CD25++ and CD45RACD25+++ cells were functionally suppressive in co-culture experiments with TCR-activated CD25CD45RA+CD4+ responder T cells.  Thus CD45RA+FoxP3lowCD25++ cells and CD45RAFoxP3highCD25+++ cells were denoted as naïve/resting and effector/activated TREGs, respectively.  CD45RAFoxP3low cells in contrast, are likely a heterogeneous mixture of cells and include some cells able to produce IFNg, IL-17, and IL-2 upon PMA+ ionomycin stimulation.  Because dividing effector T cells are able to transiently express FoxP3 at low levels, these cells are likely to be contained in the CD45RAFoxP3low population.  Thus, when using CD25 or FoxP3 to identify TREGs by flow cytometry, CD45RA should be included, and care must be taken with the gating strategies.

Treg Identification FoxP3 CD25 CD45RA resized 600

CD25 in combination with TNFR2 and/or the lack of expression of CD127 have been shown to identify FoxP3+ TREGs that are highly suppressive even in CD25low populations and thus may be excellent markers in particular for FACS sorting of TREGs for functional analyses wherein FoxP3 cannot be utilized as a selection marker.

Several other markers have been used to delineate different populations of TREGs.  The intracellular inhibitory receptor CTLA4, the co-stimulatory receptor ICOS, and the MHC class II cell surface receptor HLA-DR, are co-expressed with FoxP3 in the CD45RAFoxP3high TREG population and may be utilized as specific markers of that population.

Depending on the assay conditions, additional markers may be used to identify TREGs.  LAP, CD121a, and CD121b have been noted as highly specific markers of TREGs but are not expressed in the resting state, becoming transiently induced under assay conditions utilizing TCR stimulation.

This is by no means an exhaustive list of markers that have been used to identify human TREGs in their various functional subsets and states.  The 2011 review in Int Immunopharmacol. by Chen et. al. discusses the usage of these and other markers including CCR6, LAG-3, GARP, CD103, CD39, and CD49d.

In summary, there are multiple combinations of markers that can be used to identify functionally different TREG populations within human PBMC.  The selection of these markers should be considered in the context of the assay type being done and the questions being asked about these heterogeneous populations of cells.

Further Reading:

Regulatory T cells: mechanisms of differentiation and function.  Josefowicz SZ, Lu LF, Rudensky AY.  Annu Rev Immunol. 2012;30:531-64.

Foxp3+ regulatory T cells: differentiation, specification, subphenotypes.  Feuerer M, Hill JA, Mathis D, Benoist C. Nat Immunol. 2009 Jul;10(7):689-95.

Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor.  Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, Parizot C, Taflin C, Heike T, Valeyre D, Mathian A, Nakahata T, Yamaguchi T, Nomura T, Ono M, Amoura Z, Gorochov G, Sakaguchi S.  Immunity. 2009 Jun 19;30(6):899-911.

Resolving the identity myth: key markers of functional CD4+FoxP3+ regulatory T cells.  Chen X, Oppenheim JJ. Int Immunopharmacol. 2011 Oct;11(10):1489-96.

A peripheral circulating compartment of natural naive CD4 Tregs. D. Valmori, A. Merlo, N.E. Souleimanian, C.S. Hesdorffer, M. Ayyoub.  J. Clin. Invest., 115 (2005), pp. 1953–1962.

Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production.  Allan SE, Crome SQ, Crellin NK, Passerini L, Steiner TS, Bacchetta R, Roncarolo MG, Levings MK. Int Immunol. 2007 Apr;19(4):345-54.

CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells.   W. Liu, A.L. Putnam, Z. Xu-Yu, G.L. Szot, M.R. Lee, S. Zhu, P.A. Gottlieb, P. Kapranov, T.R. Gingeras, B. Fazekas de St Groth et al.  J. Exp. Med., 203 (2006), pp. 1701–1711

Co-expression of TNFR2 and CD25 identifies more of the functional CD4+FOXP3+ regulatory T cells in human peripheral blood.  Chen X, Subleski JJ, Hamano R, Howard OM, Wiltrout RH, Oppenheim JJ.  Eur J Immunol. 2010 Apr;40(4):1099-106.

Types of immune cells present in human PBMC

When peripheral whole blood is drawn for human immune system studies, it is often processed to remove red blood cells by density gradient centrifugation. Most commonly this method uses Ficoll Paque, a solution of high molecular weight sucrose polymers, a product of GE Healthcare Ltd.  Ficoll separates whole blood into two fractions above and below the density of 1.077g/ml.

Peripheral blood mononuclear cells (PBMC) are the populations of immune cells that remain at the less dense, upper interface of the Ficoll layer, often referred to as the buffy coat and are the cells collected when the  Ficoll fractionation method is used.

Erythrocytes (red blood cells) and polymorphonuclear cells (PMNs) which include neutrophils and eosinophils are generally removed during this fractionation as they are denser then 1.077g/ml.  Basophils, however can be greater or less dense then 1.077g/ml and thus may be present to a small degree in the less dense PBMC fraction.

PBMCs include lymphocytes (T cellsB cells, and NK cells), monocytes, and dendritic cells.  In humans, the frequencies of these populations vary across individuals.  In my experience as well as that of others, lymphocytes are typically in the range of 70 – 90% of PBMCs, monocytes range from 10 – 30% of PBMCs, while dendritic cells are rare, being only 1 – 2% of PBMCs.  The frequencies of cell types within the lymphocyte population include 70 – 85% CD3+ T cells (45 – 70% of PBMC), 5 – 20% B cells (up to 15% of PBMC), and 5 – 20% NK cells (up to 15% of PBMC).

The CD3+ compartment is composed of CD4 (25 – 60% of PBMC) and CD8 T cells (5 – 30% of PBMC), in a roughly 2:1 ratio.  Both CD4 and CD8 T cells can be further subsetted into naïve, and the antigen-experienced central memory, effector memory, and effector subtypes that exist in resting or activated states.  Multiple markers can be used to identify these compartments to varying similarities and thus the frequencies reported by people using different markers may vary.

CD4 T cells are known as helper T cells and can be further classified into various functional subtypes based on the expression profiles of specific cytokines, surface markers, or transcription factors.  These include regulatory T cells, TH1, TH2, and TH17 cells as well as other described subpopulations such as TH9, follicular helper, and TR1 types.  These classifications however will certainly become more complex in the future, as recently the cytotoxic CD8 T cell compartment has been to shown to be extremely heterogenous in marker expression and function and may be comprised of roughly 200 functional phenotypes.

Dendritic cells 2

Circulating B cells include transitional, naïve, and memory subtypes as well as plasmablasts, all of which can be found at varying populations in peripheral blood.  Circulating dendritic cells include plasmacytoid dendritic cells as well as myeloid derived dendritic cells.  Circulating monocytes have been described as either being classical monocytes or nonclassical CD16+ proinflammatory monocytes, which comprise up to 10% of the monocytes in peripheral blood and have unique functions compared with classical monocytes.

Human immune system studies rely heavily on the phenotypic and functional assessments of PBMCs.  In order to take advantage of PBMCs for human immune studies, it is important to know what populations are represented in peripheral blood and how PBMC populations differ in distribution and function from tissue immune cells.  Finally it is critical to become familiar with the identifying surface and intracellular markers and the types of assays best suited for human PBMC studies.  The markers most suitable for identification of the major immune populations in human PBMC using flow cytometry will be the topic of the next blog.