Natural Killer Cell subtypes and markers in human PBMC

Natural killer cellsNatural Killer (NK) cells are a cytotoxic innate immune lymphocyte cell type.  In humans, NK cells comprise up to 15% of peripheral blood mononuclear cells (PBMC), and 5-20% of the PBMC lymphocyte population.  Several subtypes of NK cells exist in humans.  In this post, I will discuss phenotypic properties and markers of NK subtypes present in human PBMC.

Three subtypes of NK cells are recognized: CD56dim CD16+, CD56brightCD16+/- and CD56 CD16+ NK cells. The CD56dim CD16+ and CD56brightCD16+/- subsets are best studied and are phenotypically classified as a more cytotoxic and a more cytokine producing subset of NK cells, respectively.  NK cell activation is mediated by the balance between engagement of activating receptors including NKp46, NKp30, NKp44, NKG2D, CD16, 2B4, NKp80, and DNAM-1, and HLA-I binding inhibitory receptors including killer immunoglobulin-like receptors (KIRs), LIR1/ILT2 and NKG2A/CD94.  NK cells can also be activated in response to cytokines such as IL-2, IL-12, IL-15, and IL-18.

CD56dim CD16+ NK cells:  This subtype comprises the majority, up to 90%, of PBMC NK cells and is considered the most cytotoxic subset.  CD16 is the FCγ receptor III, and can thus bind the FC portion of IgG antibodies and mediate antibody dependant cell-mediated cytotoxicity (ADCC) of antibody-bound target cells.  Expression of inhibitory receptors differs among NK subsets, and this subset exhibits lower expression of KIRs and ILT2 but higher expression of NKG2A/CD94 compared with CD56bright NK cells. Expression of granzyme B and perforin is also high in this subset compared with CD56bright NK cells.  A recent report by De Maria et. al, demonstrated that this subset does in fact robustly produce cytokines including IFNγ early after activation.

CD56brightCD16+/- NK cells: This subtype comprises up to 10% of NK cells in PBMC, but is the major NK subtype in tissues and secondary lymphoid organs.  This subset is conventionally known as the cytokine producing subset of NK cells, and rapidly produces cytokines and chemokines including IFNγ, TNFα, GM-CSF, and RANTES after activation.

Interestingly, in HIV-viremic individuals, a third CD56 CD16+ NK population is significantly expanded in PBMC comprising between 20-55% of NK cells.  This population in healthy individuals and aviremic HIV-infected individuals is rare, under 10% of total NK cells.  Compared with CD56+ NK cells, the CD56 CD16+ NK cells from HIV-viremic patients exhibited lower expression of activating receptors NKp46, NKp30, and NKp44, lower cytotoxic activity, higher expression levels of inhibitory receptors, and lower expression levels of cytokines including IFNγ, TNFα, and GM-CSF.  This subset is also expanded in individuals with chronic HCV infection.  Thus, the expansion of this poorly functional NK subset is likely clinically relevant in chronic viral disease.

In summary, these NK populations can be differentiated by expression of CD16 and CD56.  Of note, NKT (natural killer-like T) cells can also express these markers along with CD3.  Thus, to differentiate these cells from NKT cells, the inclusion of CD3 as a cell identification marker is critical in analysis of these cells by flow cytometry or other methods.


Further Reading:

CD56 negative NK cells: origin, function, and role in chronic viral disease.  Björkström NK, Ljunggren HG, Sandberg JK. Trends Immunol. 2010 Nov;31(11):401-6.

The biology of human natural killer-cell subsets. Cooper MA, Fehniger TA, Caligiuri MA. (2001) Trends Immunol 22: 633–640.

Natural killer cell distribution and trafficking in human tissues.  Carrega P, Ferlazzo G. Front Immunol. 2012;3:347.

Revisiting human natural killer cell subset function revealed cytolytic CD56(dim)CD16+ NK cells as rapid producers of abundant IFN-gamma on activation.  De Maria A, Bozzano F, Cantoni C, Moretta L. Proc Natl Acad Sci U S A. 2011 Jan 11;108(2):728-32.

Natural killer cells in HIV-1 infection: dichotomous effects of viremia on inhibitory and activating receptors and their functional correlates.  Mavilio D, Benjamin J, Daucher M, Lombardo G, Kottilil S, Planta MA, Marcenaro E, Bottino C, Moretta L, Moretta A, Fauci AS. Proc Natl Acad Sci U S A. 2003 Dec 9;100(25):15011-6.

Characterization of CD56−/CD16+ natural killer (NK) cells: a highly dysfunctional NK subset expanded in HIV-infected viremic individuals. Mavilio D, Lombardo G, Benjamin J, Kim D, Follman D, et al.. (2005) Proc Natl Acad Sci U S A. 102: 2886–2891.

Generation of CD4+ Th1 cells from human PBMC

CD4+ T helper type 1 (TH1) cells are the effector T cell population that governs cell mediated immune responses against intracellular pathogens including viruses and intracellular bacteria.  TH1 cells mediate their effect by secreting cytokines such as interferon-gamma (IFNγ) and IL-2, and express cell surface markers including CXCR3 and CCR5 and the characteristic TH1 master transcription factor T-bet (TBX21) which can also be used for detection of TH1cells by flow cytometry, as discussed in a previous blog post.

Differentiation of naïve human CD4+ T cells down the TH1 pathway involves cytokines such as IL-12 which activates STAT4, and induces expression of IFNγ and T-bet.  As such, in vitro protocols differentiating peripheral blood mononuclear cells (PBMC)-derived naïve CD4+ T cells into TH1 cells involves incubation with IL-12 in the context of T cell activation through the T cell receptor (TCR) complex.

In my experience, TH1 cells are by far the easiest CD4+ helper T cell population to generate in vitro.  In order to generate TH1 cells from human PBMC, naïve CD4+ T cells must first be isolated.  Multiple methods of naïve CD4+ T cell isolation can be utilized, and magnetic bead- based methods are common and easy methods.  Companies such as Miltenyi Biotech and Stem Cell Technologies offer kits for isolation of untouched naïve CD4+ T cells from PBMC by negative isolation methodologies.

Following isolation, naïve CD4+ T cells are activated through the TCR complex.  Tissue culture plates can be coated with anti-CD3 (OKT1) and anti-CD28 antibodies in PBS prior to culture.  Alternatively, naïve CD4+ T cells can be cultured with Dynal CD3/CD28 T Cell Expander Dynabeads (Life Technologies) at a 1 bead per cell ratio.  A third alternative involves coating tissue culture plates with anti-CD3 alone and obtaining CD28 co-stimulation by the addition of autologous monocytes isolated from PBMCs into the culture.

To generate TH1 cells, recombinant human IL-12 is added alone, or at a lower dose in combination with anti-IL-4 blocking antibodies to inhibit the counteractive effects of IL-4 and TH2 pathways on TH1 cell polarization.  Finally recombinant human IL-2 is added to promote T cell proliferation.  Media and cytokines/blocking antibodies are refreshed every two to three days depending on the cell density, and as the cells expand the time to refresh the media shortens.

Lymphocyte activationTH1 cells can be generated and assayed for functions including IFNγ expression in as few as three days.  If long term or clonal T cells assays are of interest, cells can be expanded in the presence of IL-2 for 2-3 weeks following single cell cloning.  As previously discussed, TH1 cells can be identified by IFNγ expression following a 4-6 hour incubation with TCR activation by plate bound anti-CD3 plus anti-CD28, CD3/CD28 Dynabeads, or PMA/ionomycin in the presence of brefeldin-A.  Cells are then fixed, permeabilized, and stained for cell surface markers and intracellular IFNγ.

Finally, as a comparison, tandem experiments can be run in which naïve CD4+ T cells are maintained under non-polarizing (TH0) conditions.  For this, often no cytokines aside from IL-2 are added.  However the addition of anti-IL-12 and anti-IL-4 may be necessary to inhibit any cells from differentiating down TH1 or TH2 pathways by production of these cytokines by the T cells themselves.

In conclusion, generation of CD4+ TH1cells from human PBMC is a relatively simple and straightforward protocol, and very high percentages of TH1cells can be obtained through optimized protocols.


Further Reading:

Differentiation of effector CD4 T cell populations (*).  Zhu J, Yamane H, Paul WE.  Annu Rev Immunol. 2010;28:445-89.

Memory and flexibility of cytokine gene expression as separable properties of human T(H)1 and T(H)2 lymphocytes.  Messi M, Giacchetto I, Nagata K, Lanzavecchia A, Natoli G, Sallusto F.  Nat Immunol. 2003 Jan;4(1):78-86.

A critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses.  Volpe E, Servant N, Zollinger R, Bogiatzi SI, Hupé P, Barillot E, Soumelis V. Nat Immunol. 2008 Jun;9(6):650-7.

Identification of CD8+ TC1, TC2, and TC17 populations in human PBMC

Human peripheral blood mononuclear cells (PBMC) are composed of heterogeneous populations of various immune cell types.  CD4+ and CD8+ T cells are known to exist in various functional and differentiated states.  Following antigen experience, naïve T cells are thought to progressively differentiate along a path through central memory, effector memory, and terminally differentiated effector states. Markers for differentiating PBMC T cells into these subtypes using multiparametric flow cytometry include: CD3, CD4, CD8, CD45RA or CD45RO, and CCR7 or CD62L.

The cytokine milieu T cells are exposed to during antigen encounter directs differentiation into various subtypes that exhibit unique functional properties and gene expression programs including cytokines, transcription factors, and surface markers.  This is true for both CD4+ and CD8+ T cells. In a previous post, I discussed various markers that can be utilized by flow cytometry to identify CD4+ TH1, TH2, and TH17 populations in human PBMC.

CD8+ T cells can also differentiate into unique subsets similar to TH1, TH2, and TH17 CD4+ T cells.  The CD8+ versions of these subsets are referred to as TC1, TC2, and TC17 CD8+ T cells, respectively, and are defined by expression of the same characteristic cytokines as their CD4+ counterparts.

As previously discussed, expression of subset-specific surface markers is easily determined by flow cytometry. Identification of intracellular cytokine production in T cells can be assessed following 4-6 hours of TCR stimulation with anti-CD3 and anti-CD28 antibodies or the combination of Phorbol 12-Myristate 13-Acetate (PMA) and ionomycin in the presence of brefeldin A or monensin.  The cells are then fixed and permeabilized with buffers such as BD Biosciences’ Cytofix Cytoperm buffer set followed by antibody staining for cytokine expression and flow cytometry.

Like CD4+ TH1 cells, CD8+ TC1 cells characteristically produce IFNγ.  This population is by far the most common cytokine-producing CD8+ cell subset, and is very easy to identify using intracellular staining for IFNγ.

As with CD4+ TH2 cells, CD8+ TC2 cells can be identified by expression of IL-4, IL-5, and IL-13Cosmi et. al, found that the surface marker CRTH2 was a robust marker for identification of CD8+ and CD4+ cells producing IL-4, IL-5, and IL-13 expression but not IFNγ.  Expression of chemokine receptors CCR3 and CCR4 however, did not exclude IFNγ-producing cells.  Because expression of IL-4, IL-5, and IL-13 can be difficult to detect, CRTH2 may be the easiest of these markers for TC2 identification in human PBMC.

CD8+ TC17 cells are characterized by expression of the cytokine IL-17.  Expression of the chemokine receptors CCR5 and CCR6 were shown to enrich for IL-17 producing CD8+ cells.  However CCR5 and CCR6 expression are also associated with TC1 cells, and thus may not be useful to differentiate between these subsets.

CD8 Tc1 Tc2 Tc17 PMA resized 600

Figure: Expression of IFNγ, IL-17, and CRTH2 in CD8+ T cells from PBMC stimulated with for 4 hours with PMA/ionomycin.

In my own studies, I have utilized TCR or PMA/ionomycin stimulation of PBMCs to successfully identify IFNγ (TC1) and IL-17 (TC17) expressing cells, and CRTH2 expression to identify TC2 cells, as these may be the most robust markers for identification of these unique CD8+ T cell populations. Note also that these same markers reliably detect CD4+ TH1, TH17, and TH2 cells, respectively. Thus, these markers are useful to quantitate and study the function of both CD8+ and CD4+ subsets in human PBMC.


Additional Reading

Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles.  Croft M, Carter L, Swain SL, Dutton RW. J Exp Med. 1994 Nov 1;180(5):1715-28.

CRTH2 is the most reliable marker for the detection of circulating human type 2 Th and type 2 T cytotoxic cells in health and disease.  Cosmi L, Annunziato F, Galli MIG, Maggi RME, Nagata K, Romagnani S.  Eur J Immunol. 2000 Oct;30(10):2972-9.

Cutting edge: Phenotypic characterization and differentiation of human CD8+ T cells producing IL-17.  Kondo T, Takata H, Matsuki F, Takiguchi M. J Immunol. 2009 Feb 15;182(4):1794-8.

Functional expression of chemokine receptor CCR6 on human effector memory CD8+ T cells.  Kondo T, Takata H, Takiguchi M. Eur J Immunol. 2007 Jan;37(1):54-65.

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*

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.