Category: PBMC Basics

CXCR5 is a chemokine receptor expressed by and used to identity human CD4⁺ follicular helper T cells (T_FH).  T_FH cells, as their name implies, promote the differentiation and survival of memory and plasma B cells in the B cell follicular and germinal center regions of secondary lymphoid organs. CXCR5⁺ T_FH-like central memory CD4⁺ T cells (CD4⁺ T_CM) also circulate in peripheral blood and can be detected among human peripheral blood mononuclear cells (PBMC).  CXCR5⁺ cells comprise 20-25% of CD4⁺ T_CM cells in human PBMC.  However, what are the identifying markers and functional differences of CXCR5⁺ vs. CXCR5^– CD4⁺ T cells from human PBMC and the prototypical CXCR5⁺ T_FH cells in secondary lymphoid organs?

I have previously discussed markers that can be used for identification of human CD4⁺ T_H1, T_H2, and T_H17 T cell helper subsets as well as CD4⁺ FoxP3⁺ regulatory T cells.  CXCR5 can be upregulated transiently on activated T cells, however a subset of PBMC T cells constitutively express CXCR5, indicating this is a uniquely functioning subset identifiable by this marker using flow cytometry or other methods.  PBMC CXCR5⁺ CD4⁺ T_CM cells exhibit many but not all features of T_FH cells present in secondary lymphoid organs, and thus may be the circulating memory counterpart of T_FH cells.

CXCL13, the chemokine ligand for CXCR5, is highly expressed in B cell follicles and likely plays an important role in recruitment of CXCR5⁺ T_FH cells to B cell zones.  Expression of ICOS by T_FH cells has been demonstrated to be essential for their function in B cell help.  Additionally, T_FH cells are higher expressers of CXCL13, as well as IL-21, IL-10, Bcl-6, and PD-1 than other helper T cell subsets.

Studies by Chevalier et al, and Morita et. al. compared the functional properties of CXCR5^– and CXCR5⁺ CD4⁺ T_CM cells from human PBMC.  PBMC CXCR5⁺ CD4⁺ cells are resting central memory cells in phenotype, being CD45RA^–, CCR7⁺ and CD62L⁺, but not expressing activated T_FH markers such as ICOS and CD69.  Upon stimulation, CXCR5⁺ CD4⁺ T_CM promote significantly higher B cell plasmablast differentiation and Ig secretion than CXCR5^– CD4⁺ T_CM cells, attributable to enhanced expression of ICOS and IL-10.  However, Bcl-6 expression was not found to be different between these PBMC subsets, and conclusions for expression levels and role of IL-21 were contradictory between these studies.

The question of whether PBMC CXCR5⁺ CD4⁺ T_CM are distinct from T_H1, T_H2, and T_H17 T cells was addressed by these studies as well. While Chevalier et al. found that CXCR5⁺ T_CM cells were more non-polarized and secreted comparatively lower levels of cytokines associated with T_H1, T_H2, and T_H17 T cells, Morita et. al, identified T_H1, T_H2, and T_H17 T cells within CXCR5⁺ compartment, albeit at somewhat different frequencies than CXCR5^– cells. Thus PBMC CXCR5⁺ CD4⁺ T_CM are a heterogenous subset with features of both T_FH cells and the various T_H1, T_H2, and T_H17 subsets.  Further interrogation of the functions of these populations are needed.

PBMC CXCR5⁺ CD4⁺ cells have been identified as a highly relevant population to study in the context of vaccination and human disease.  Patients with systemic lupus erythematosus (SLE) have higher percentages of circulating CD4⁺CXCR5⁺ ICOS⁺ cells.  Patients with autoimmune juvenile dermatomyositis (JDM) were found to have an altered CXCR5⁺ compartment where the overall frequency of CXCR5⁺ cells was not different, but the ratio of T_H2 and T_H17 to T_H1 cells within the CXCR5⁺ population was enhanced and associated with disease activity.

In the vaccine setting, emergence of a population of circulating ICOS⁺CXCR3⁺CXCR5⁺CD4⁺ T cells was found in individuals 7 days after influenza vaccination, and correlated with increased antibody titers and B cell plasmablasts. These cells could also induce plasma cell differentiation in vitro and  thus are important in the development of vaccine elicited protective antibody responses.

In conclusion, PBMC CXCR5⁺ CD4⁺ T cells are an important cellular subset to study in the context of human disease.  These cells are likely the circulating memory component of T_FH cells, and alterations in frequencies and functions are associated with various human diseases and protective antibody responses following vaccination.

Further Reading:

Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion.  Morita R, Schmitt N, Bentebibel SE, Ranganathan R, Bourdery L, Zurawski G, Foucat E, Dullaers M, Oh S, Sabzghabaei N, Lavecchio EM, Punaro M, Pascual V, Banchereau J, Ueno H. Immunity. 2011 Jan 28;34(1):108-21.

CXCR5 expressing human central memory CD4 T cells and their relevance for humoral immune responses.  Chevalier N, Jarrossay D, Ho E, Avery DT, Ma CS, Yu D, Sallusto F, Tangye SG, Mackay CR. J Immunol. 2011 May 15;186(10):5556-68.

Expansion of circulating T cells resembling follicular helper T cells is a fixed phenotype that identifies a subset of severe systemic lupus erythematosus. N. Simpson, P.A. Gatenby, A. Wilson, S. Malik, D.A. Fulcher, S.G. Tangye, H. Manku, T.J. Vyse, G. Roncador, G.A. Huttley et al.  Arthritis Rheum., 62 (2010), pp. 234–244.

Induction of ICOS+CXCR3+CXCR5+ TH Cells Correlates with Antibody Responses to Influenza Vaccination.  Bentebibel S. E. et al.  Sci Transl Med. 13 March 2013: Vol. 5, Issue 176, p. 176ra32.

Photo credit: wellcome images / Foter.com / CC BY-NC-ND

Natural 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: CD56^dim CD16⁺, CD56^brightCD16^+/- and CD56^– CD16⁺ NK cells. The CD56^dim CD16⁺ and CD56^brightCD16^+/- 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.

CD56^dim 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 CD56^bright NK cells. Expression of granzyme B and perforin is also high in this subset compared with CD56^bright 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.

CD56^brightCD16^+/- 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.

CD4⁺ T helper type 1 (T_H1) cells are the effector T cell population that governs cell mediated immune responses against intracellular pathogens including viruses and intracellular bacteria.  T_H1 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 T_H1 master transcription factor T-bet (TBX21) which can also be used for detection of T_H1cells by flow cytometry, as discussed in a previous blog post.

Differentiation of naïve human CD4⁺ T cells down the T_H1 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 T_H1 cells involves incubation with IL-12 in the context of T cell activation through the T cell receptor (TCR) complex.

In my experience, T_H1 cells are by far the easiest CD4⁺ helper T cell population to generate in vitro.  In order to generate T_H1 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 T_H1 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 T_H2 pathways on T_H1 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.

T_H1 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, T_H1 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 (T_H0) 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 T_H1 or T_H2 pathways by production of these cytokines by the T cells themselves.

In conclusion, generation of CD4⁺ T_H1cells from human PBMC is a relatively simple and straightforward protocol, and very high percentages of T_H1cells 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.

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+ T_H1, T_H2, and T_H17 populations in human PBMC.

CD8⁺ T cells can also differentiate into unique subsets similar to T_H1, T_H2, and T_H17 CD4+ T cells.  The CD8⁺ versions of these subsets are referred to as T_C1, T_C2, and T_C17 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⁺ T_H1 cells, CD8⁺ T_C1 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⁺ T_H2 cells, CD8⁺ T_C2 cells can be identified by expression of IL-4, IL-5, and IL-13.  Cosmi 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 T_C2 identification in human PBMC.

CD8⁺ T_C17 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 T_C1 cells, and thus may not be useful to differentiate between these subsets.

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 (T_C1) and IL-17 (T_C17) expressing cells, and CRTH2 expression to identify T_C2 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⁺ T_H1, T_H17, and T_H2 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.

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.

CD95 (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 http://en.wikipedia.org/wiki/Fas_ligand*