Co-Staining for Pro5® Pentamers and Intracellular Cytokines

Co-staining CD8+ T cells with Pro5® MHC Pentamers and antibodies against intracellular cytokines has now become a routine procedure in many laboratories. This technique not only enables the frequency of antigen-specific T cells to be determined but also their effector function.

Production of cytokines plays an important role in the immune response. Examples include the induction of many anti-viral proteins by IFN gamma, the induction of T cell proliferation by IL-2 and the inhibition of viral gene expression and replication by TNF alpha. Cytokines are not preformed factors, instead they are rapidly produced upon relevant stimulation. Intracellular cytokine staining relies upon the stimulation of T cells in the presence of an inhibitor of protein transport thus retaining the produced cytokines inside the cell.

Cellular activation to trigger cytokine production generally results in down-regulation of the T cell receptor. For this reason, Pentamer staining is carried out prior to activation to ensure a good level of staining. The Pentamers may be internalized with the T cell receptor during this period, but can still be detected in permeabilized cells.

To analyze the effector function of antigen-specific T cells, the cells are first stained with Pentamer, then stimulated with antigen. This is followed by staining with antibodies specific for extracellular epitopes (such as CD8), then by membrane permeabilization and intracellular cytokine staining (ICS).


Example Staining Data Example Staining Protocols
Customer Case Studies Customer Publications


Example Staining Data

(1) Gating Strategy

Create a 2D density plot showing FSC vs. SSC. Draw a tight region around the lymphocyte population (R1)

Create a second density plot, gated only on events encircled within R1, showing CD8 staining on the x-axis and Pentamer staining on the y-axis. Draw a region around all of the CD8-positive cells (R2).

Create a third density plot, gated on both R1 and R2, showing cytokine staining on the x-axis and Pentamer staining on the y-axis. Draw a quadrant that clearly separates positive cells from negative cells on both axes, and use quadrant statistics to obtain quantitative information about the frequency of antigen-specific cytokine-positive cytotoxic T lymphocytes.

Data plots illustrating example gating strategy


(2) Example Figures

IFN gamma versus Pentamer staining of live lymphocytes

The figure above illustrates IFN gamma versus Pentamer staining of live lymphocytes. PBMCs were incubated with either a negative control (non-specific) Pentamer (A*02:01/GLCTLVAML (EBV) or one of two Pentamers specific for the cells of interest (B*08:01/RAKFKQLLEBV (EBV), or B*08:01/FLRGRAYGL (EBV), then stimulated with the appropriate B*08:01/EBV peptide for 15 hours using the protocol detailed in the PDF link below. Fixation, permeabilization and staining for IFN gamma were also carried out exactly as detailed in the protocol.

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Protocols for co-staining with Pentamers and intracellular cytokines

Links to protocols applicable for MHC Pentamer co-staining with anti-IFN gamma, TNF alpha, MIP-1 beta, or IL-2. Note that certain cytokines such as IL-4 and IL-10 are difficult to detect by intracellular staining methods. In addition to this, frequencies of Pentamer-positive cells producing these cytokines may also be extremely low.

Human intracellular cytokine and Pentamer staining protocol (PDF)

Murine intracellular cytokine and Pentamer staining protocol (PDF)


Intracellular Cytokine Staining as an Outsourced Service

ProImmune offers rapid and reliable flow cytometric detection of human IFN gamma, IL-2 and TNF alpha cytokine production in CD4+ and CD8+ T cells through its experienced applications team. By outsourcing your intracellular cytokine staining experiments to ProImmune, you take advantage of our technical proficiency and save time by passing collection and analysis of data to our experts.

Further information about our Intracellular Cytokine Staining Service


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Case Study 1:

Costaining for Pro5® Pentamer and IFN gamma illustrates differential presentation of CD8+ T cell epitopes

Tellam, J. et al. (2006). Influence of translation efficiency of homologous viral proteins on the endogenous presentation of CD8+ T cell epitopes J. Exp. Med. 204(3): 525-32. [PubMedID: 17312009]

Judy Tellam, from the group of Professor Rajiv Khanna, at the Queensland Institute of Medical Research, and Mark Fogg from Harvard Medical School used a Pro5® MHC Class I Pentamer and intracellular cytokine staining in their study to show how protein translation affects the molecular dynamics of antigen presentation.


The study compared the immune responses to EBV encoded nuclear antigen, EBNA1, with and without its internal Gly-Ala repeat sequence. These two versions of the protein have significantly different protein translation efficiencies. A combination of the B*08:01/FLRGRAYGL Pro5® Pentamer and costaining for interferon gamma expression was used to assess the activation of FLR-specific T cells that were stimulated with lymphoblastoid cell lines (LCLs) expressing the two different EBNA1 proteins.

A significant increase in Pentamer-positive and interferon gamma-expressing T cells was observed when EBV EBNA1-specific T cells were stimulated with LCLs that were transduced with an adenoviral construct carrying the EBNA1 antigen without the Gly-Ala repeat (EBNA1-GA) compared to those stimulated with LCLs transduced with the full-length EBNA1.


Case Study 2:

Study using HIV-specific Pro5® MHC Class I Pentamers with intracellular cytokine staining provides useful data for the development of T cell vaccine

Almeida, J.R. et al. (2007). Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J Experimental Medicine 204: 2473-2485. [PubMedID:17893201]

Almeida et al. carried out an in-depth analysis of the MHC Class I B*27:05/KRWIILGLNK (KK10) HIV-1 epitope. Previous studies have shown that the presence of B27-KK10 epitope-specific T cells confers some protection against progression of the disease in HIV-positive individuals.

Pro5® Pentamers specific to several HIV-1 epitopes were used to identify the antigen-specific cells, which were then further analyzed by flow cytometry to determine their phenotypic characteristics. Additionally, the capacity of B27-KK10-specific CD8+ T cells to produce effector cytokines and to release cytotoxic factors upon antigenic stimulation was assessed with a B*27:05/KRWIILGLNK (KK10)-specific Pentamer and intracellular cytokine staining in order to provide a picture of CD8+ T cell functional quality.

Interestingly, the B27/KK10 CD8+ T cell population showed a significantly higher proportion of cells producing effector cytokines and cytotoxic factors compared with other HIV-specific CD8+ T cells, suggesting that these cells are highly functional. This polyfunctional capability may contribute in part to the protective characteristics and better disease control associated with this epitope. The authors commented that combined data from the study might aid in the development of successful T cell vaccines.

A representative example of simultaneous multifunctional assessment of B27-KK10-specific CD8+ T cells

A representative example of simultaneous multifunctional assessment of B27-KK10-specific CD8+ T cells using nine-colour flow cytometry. Cells were stimulated for 6 h in the presence of cognate peptide before intracellular cytokine staining. Percentages of cells in the different quadrants are shown. Plots are gated on CD3+CD8+ cells. Copyright Rockefeller University Press 2007.

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Publications citing the use of Pro5® MHC Pentamers in combination with ICS

Almeida, J. et al. (2007). Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J. Exp. Med. 204: 2473-85. [PubMedID: 17893201]

B*27:05/KRWIILGLNK (HIV-1 gag p24)

A*03:01/QVPLRPMTYK (HIV-1 nef)

A*03:01/RLRPGGKKK (HIV-1 gag p17)

B*07:02/GPGMKARVL (HIV-1 gag)

Bailey, JR. et al. (2008). Transmission of human immunodeficiency virus type I from a patient who developed AIDS to an elite suppressor. J Virol. 82: 7395-7410. [PubMedID: 18495769]

B*27:05/KRWIILGLNK (HIV-1 gag p24)

Caccamo, N. et al. (2006). Phenotypical and functional analysis of memory and effector human CD8 T cells specific for mycobacterial antigens.  J. Immunol. 177: 1780-1785. [PubMedID: 16849488]

A*02:01/GLPVEYLQV (Mycobacterium bovis antigen 85-A)

Casado, JG. et al. (2009) Correlation of effector function with phenotype and cell division after in vitro differentiation of naive MART-1-specific CD8+ T cells. International Immunolgy. 21: 53-62. [PubMedID: 19050104]

A*02:01/ELAGIGILTV (MelanA/MART-1)

Fousteri, G. et al. (2009). CD103 is dispensable for anti-viral immunity and autoimmunity in a mouse model of virally-induced autoimmune diabetes. J. Autoimmunity. 32: 70-77. [PubMedID: 19162441]



Gandhi, M.K. et al.  (2007). Galectin-1 mediated suppression of EBV-specific T-cell immunity in classical Hodgkin-s lymphoma. Blood 110: 1326-9. [PubMedID: 17438085]


Grolleau-Julius, A. et al. (2008). Impaired dendritic cell function in aging leads to defective antitumor immunity. Cancer Res. 68: 6341-6349. [PubMedID: 18676859]

H-2Kb/SIINFEKL (Ovalbumin)

Karlsson, H. et al.  (2008). Mesenchymal stem cells exert differential effects on alloantigen and virus-specific T cell responses. Blood 112: 532-541. PubMedID: 18445691]

A*02:01/NLVPMVATV (HCMV pp65)






Radford, KJ. et al. (2006). CD11c+ blood dendritic cells induce antigen-specific cytotoxic T lymphocytes with similar efficiency compared to monocyte-derived dendritic cells despite higher levels of MHC class I expression. J Immunother. 29: 596-605. [PubMedID: 17063122]

A*02:01/ELAGIGILTV (MelanA/MART-1)

A*02:01/GILGFVFTL (Influenza MP)

Rawson, P.M. et al. (2007). Cross-presentation of caspase-cleaved apoptotic self antigens in HIV infection. Nat. Med. 13: 1431-1439. [PubMedID: 18026114]

A*02:01/LLQDSVDFSL (Vimentin)

A*02:01/QLFNHTMFI (Non-muscle myosin)

A*02:01/VLMIKALEL (Non-muscle myosin)

A*02:01/FLGMESCGI (Actin)

Smith, C et al. (2009). Acquisition of polyfunctionality by Epstein-Barr virus-specific CD8+ T cells correlates with increased resistance to galectin-1 mediated suppression.  J Virol. 83: 6192-6198. [PubMedID: 19357166]



Suneetha, PV. et al. (2009) Effect of peptide pools on effector functions of antigen-specific CD8+ T cells. J Immunol. Methods 342: 33-48. [PubMedID: 19135447]

A*02:01/GILGFVFTL (Influenza MP)

A*02:01/NLVPMVATV (CMV pp65)



Tellam, J. et al. (2007). Influence of translation efficiency of homologous viral proteins on the endogenous presentation of CD8+ T cell epitopes. J. Exp. Med. 204: 525-532. [PubMedID: 17312009]

B*08:01/FLRGRAYGL (EBV EBNA-3A 193-201)

Tellam, J. et al. (2008). Regulation of protein translation through mRNA structure influences MHC class I loading and T cell recognition. PNAS. 105: 9319-9324. [PubMedID: 18591662]

B*35:08/HPVGEADYFEY (EBV EBNA1407-417)

Yagi, H. et al. (2006). Induction of therapeutically relevant cytotoxic T lymphocytes in humans by percutaneous peptide immunization.Cancer. Res. 66: 10136-10144. [PubMedID: 17047078]



Zhong, J. et al. (2008). Induction of pluripotent protective immunity following immunisation with a chimeric vaccine against human cytomegalovirus. PLoS one. 3:e3256. [PubMedID: 18806877]

A*02:01/NLVPMVATV (CMV pp65)

A*01:01/VTEHDTLLY (CMV pp50)


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