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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: Cytometry A. 2014 Nov 18;87(4):289–292. doi: 10.1002/cyto.a.22590

OMIP-025: evaluation of human T- and NK-cell responses including memory and follicular helper phenotype by intracellular cytokine staining

Gemma Moncunill 1,2,*, Carlota Dobaño 2, M Juliana McElrath 1,3, Stephen C De Rosa 1,4
PMCID: PMC4454451  NIHMSID: NIHMS694613  PMID: 25407958

Purpose and Appropriate Sample Types

This panel was developed to assess antigen-specific T cells using peptide pools to various antigens of interest, although other types of antigens such as recombinant proteins or whole pathogens could be considered using different stimulation times. In addition to multiple functional markers, the panel includes differentiation markers and markers to assess follicular helper T cells and NK cells (Table 1). It was optimized using cryopreserved peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV) uninfected and HIV infected adults with known cytomegalovirus (CMV) responses and it underwent assay qualification. The panel is being used to evaluate the responses to HIV and malaria vaccine candidates in adults and children from different geographic areas.

Keywords: cytometry, human PBMC, T cells, TFH cells, NK cells, memory, intracellular cytokines

Background

Vaccines are one of the most cost-effective interventions, saving millions of lives every year. Yet, despite all the efforts invested, vaccines against complex diseases such as HIV, TB and malaria remain resistant to development. One of the main impediments is the lack of correlates of protection, whose identification would improve and accelerate the design and development of vaccine candidates for these infectious diseases.

This panel was designed starting from the OMIP-014 panel (1) to include the maximum number possible of relevant markers for vaccine immunogenicity that could lead to the identification of correlates of protection. High-level polychromatic panels are a requirement when assessing vaccine responses with a limited amount of sample, particularly when the population under study is infants and children, as is often the case in vaccine development efforts for malaria and tuberculosis (2–4).

A prerequisite of the new panel was to retain at least the same sensitivity for interferon-gamma (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α) as in OMIP-014, which is widely used by the HIV Vaccine Trials Network (HVTN). Therefore, our priority was the assessment of IFN-γ, IL-2 and TNF-α markers. Of importance, the OMIP-014 panel was validated for IFN-γ and IL-2 responses and the current panel underwent assay qualification comparing the responses with this panel (online material). After testing a few new reagents for these three cytokines that did not prove to be better than the bright and sensitive ones used in OMIP-014, we opted to utilize the same reagents for the new expanded panel. Ultimately, only these three cytokine reagents and the viability marker remained unchanged from OMIP-014; the reagents for the remainder of shared markers (CD3, CD4, CD8, CD154 and IL-4) had to be changed through the panel development process (online material). For instance, CD4 conjugated to APC-Ax750 was replaced for several reasons (online material), including suspected breakdown of the tandem fluorochrome resulting in non-specific events detected in the APC channel that was reserved for the low-frequency functional marker IL-4 in OMIP-014, and which was eventually used here for IL-21.

IL-4, a representative TH2 cytokine, is difficult to detect and the responses have been low in HIV vaccine studies conducted through the HVTN. Despite this, when combined with other functional markers in polyfunctionality analyses, polarized TH1 and TH2 CD4+ T cells have been detected for different vaccine regimens (5) and T cells co-expressing IL-4 with CD154, IFN-γ, IL-2 and TNF-α inversely correlate with infection risk in the RV144 HIV vaccine trial (unpublished data). Therefore, we decided to retain IL-4, changing the conjugated fluorochrome from APC to PerCP-Cy5.5. In the APC channel we added IL-21, a TFH immunoregulatory cytokine, which is also difficult to detect due to its low expression in circulating T cells. CD154 (CD40L) is expressed mainly on activated CD4+ T cells, is key to providing T-cell help to B cells, and is a relevant sensitive and specific marker for the detection of antigen-specific T cells upon ex vivo stimulations (6,7). Therefore, we also kept it in the new panel, although the reagent was changed to an antibody conjugated to BV605 instead of PE-Cy5 because the latter showed extensive fluorescence spreading into other channels, including those detecting functional makers. As in OMIP-014, CD154 staining was performed intracellularly (1). Although CD154 can be detected by surface staining by including the CD154 reagent during the ex vivo antigen stimulation, the usage of brefeldin A is incompatible with this method because it completely blocks CD154 surface expression (8).

To discern the memory subset of the antigen-specific T cells, the differentiation markers CCR7 and CD45RA were included. The combination of both markers allows the identification of naïve (CD45RA+CCR7+), central memory (CD45RA-CCR7+), effector memory (CD45RA-CCR7−) and terminal effector (CD45RA+CCR7−) subsets (9–11). After several tests, CCR7 conjugated to BV785 and CD45RA to APC-H7 were selected.

One of our main interests was the inclusion of markers to identify peripheral follicular helper T cells (TFH cells), a TH subset involved in germinal center reactions necessary for the development of high affinity and long-term antibody responses (12). Circulating TFH-like cells have been mostly defined as CXCR5+ CD4+ memory T cells (13,14), although different subsets of CXCR5+ CD4+ T cells have been studied including the CCR7lo PD-1hi cells, which have been described as TFH precursors that correlate with active TFH differentiation in secondary lymphoid organs and antibody responses (15). Therefore, the chemokine receptor CXCR5 and the inhibitory marker PD-1 were added in the panel conjugated to PE-eFluor610 and PE-Cy7, respectively. Additionally, TFH and TFH-like cells express inducible costimulator (ICOS) at high density in the lymphoid organs and in the periphery, respectively, when they are activated (12–15). We tested several ICOS reagents but this marker was ultimately excluded, as it did not provide satisfactory results in combination with the other makers in the panel.

Additionally, CD56 BV650 was incorporated to evaluate effector NK cell responses, since this leukocyte population has been shown to be a key producer of IFN-γ in acquired immune responses, contributing to the vaccine-induced response (16,17). The marker CD56 allows the discrimination of the two main subsets of NK cells that have different receptors and functions (18): CD56high (CD56hiCD3−) and CD56dim (CD56dimCD3−). The inclusion of CD56+ permitted also the identification of NKT-like cells defined as CD56+CD3+ cells, which are different than the CD1d-restricted invariant NK T cells (19).

Finally, to improve the specificity of the assay, a viability marker was included for the exclusion of dead cells that may non-specifically bind antibodies and contribute to background (20). CD14 was similarly included to exclude monocytes, and in contrast to OMIP-014, it is detected in the same channel as the viability marker, thus creating a dump channel.

The reagents used for this panel are listed in Table 2. Figure 1 shows an example staining profile for PBMC stimulated with staphylococcal enterotoxin B. Further developmental strategies and details for the panel may be found in the online material.

Table 2.

Reagents used for OMIP-025

SPECIFICITY CLONE FLUOROCHROME PURPOSE
CD3 UCHT1 BV570 T lineages
CD8 RPA-T8 BV711
CD4 SK3 BUV395
CXCR5 MU5UBEE PE-eFluor610 TFH
PD-1 eBioJ105 PE-Cy7
CD45RA HI100 APC H7 Memory/Differentiation
CCR7 G043H7 BV785
CD56 HCD56 BV650 NK cells, NKT-like cells
IFN-γ B27 V450 Function
IL-2 MQ1-17H12 PE
TNF-α MAb11 FITC
IL-4 MP4-25D2 PerCP-Cy5.5
IL-21 3A3-N2 APC
CD154 24–31 BV605
CD14 M5E2 BV510 Monocytes (Dump)
Live/Dead NA AViD Dead cells (Dump)
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APC, allophycocyanin; Ax, Alexa; AViD, LIVE/DEAD fixable aqua dead cell stain; BUV, brilliant ultraviolet; BV, brilliant violet; Cy, cyanine; FITC, fluorescein isothiocyanate; NK, natural killer; PE, R-phycoerythrin; PerCP, peridinin chlorophyll protein; TFH, follicular helper T cells.

Figure 1. Example of the staining and gating strategy for PBMC stimulated with staphylococcal enterotoxin B (SEB).

Figure 1

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All gates for non-functional markers were defined using fluorescence minus one (FMO) controls whereas gates for functional markers were defined using the unstimulated samples. (A) Gating hierarchy to identify NK cells, NKT-like cells, CD4+ and CD8+ T cells and TFH-like cells. Initial gating is done on FSC-H and FSC-A to discriminate singlets, followed by the exclusion of events collected during a period of time early in collection when fluctuations may occur. In this example there were no problems of fluctuations and the time gate was minimized to avoid exclusion of any events. Dead cells and monocytes are excluded by an amine reactive dye and the CD14 marker in the same dump channel. Lymphocytes are gated using FSC-A and SSC-A. Subsequent gating discriminates two subsets of NK cells by CD56 and CD3 expression (CD56dimCD3− and CD56hiCD3−) and NKT-like cells (CD56+CD3+). Within the gate of lymphocytes, CD3+ cells are identified, followed by identification of CD4+ and CD8+ T cells. Of note, NKT-like cells are not excluded from classical T cells and therefore are overlapping populations. Finally, TFH-like cells are identified as CXCR5+ CD45RA- CD4+ T cells that have a low expression of CCR7 and are PD-1+. (B) Functional markers for CD4+ and CD8+ T cells. A gate is applied for each cytokine, not taking into account the coexpression of other markers. Boolean gates are then created based on these gates to identify cells expressing different combinations of markers. (C) The expression level of CCR7 and CD45RA is examined within CD4+ and CD8+ T-cell subsets to later provide insight into the memory phenotype of the antigen specific cells.

Similarity to published OMIPs

This panel is an expansion and modification of the OMIP-014 panel (1), which now includes differentiation and TFH markers, whereas MIP-1β and CD107a were removed. In addition, it presents similarities to other OMIP panels with intracellular cytokine staining to identify antigen specific T cells: OMIP-001 (21), OMIP-008 (22), OMIP-009 (23) and OMIP-022 (24). However, our panel includes TFH markers, the NK marker CD56, and cytokine IL-21, which are not addressed in any of the other ICS OMIP panels.

Supplementary Material

OMIP025 supplmentary material

Table 1.

Summary table for application of OMIP-025

Purpose Characterization of antigen-specific T cells, TFH-like cells and NK cells
Species Human
Cell types Cryopreserved PBMC
Cross-references OMIP-014
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TFH, follicular helper T cells; NK, natural killer; PBMC, peripheral blood mononuclear cells

Acknowledgements

The authors wish to thank all the individuals enrolled in the Seattle Assay Control cohort, from which PBMC were used for optimization and testing of the panel. The authors are grateful to HVTN technicians Hannah H. Han for her help in the titrations and panel tests, and also Nathaniel Chartrand, Paul Newling, Bryce Manso, Kevin Hawkins and Terry Stewart for their help in the lab. We acknowledge Stephen Voght for his help with editing. We thank the Vaccine Immunology Workgroup (VIW) of the RTS,S immune correlates study (Mal067) for their support in developing the panel and particularly the VIW Cellular Working Group for their inputs and discussion in the panel design. We thank the James B. Pendleton Charitable Trust for their generous equipment donation.

Grant sponsors: HIV Vaccine Trials Network Laboratory Center (HVTN, National Institute of Allergy and Infectious Diseases), grant number: UM1 AI068618; Human Immunology Project Consortium (HIPC, National Institute of Allergy and Infectious Diseases), grant number: P30 AI027757; Instituto de Salud Carlos III, salary support to G.M., fellowship number: CD10/00156.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

OMIP025 supplmentary material
NIHMS694613-supplement-OMIP025_Supplementary_material.docx (8.4MB, docx)

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