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Published in final edited form as: J Immunol Methods. 2006 Apr 19;312(1-2):45–53. doi: 10.1016/j.jim.2006.02.018

Detection of macaque perforin expression and release by flow cytometry, immunohistochemistry, ELISA, and ELISpot

Bartek Zuber a,*, Máire F Quigley b, J William Critchfield c, Barbara L Shacklett c, Kristina Abel d, Christopher J Miller d, Andreas Mörner e, Staffan Paulie a, Niklas Ahlborg a, Johan K Sandberg b
PMCID: PMC11910751  NIHMSID: NIHMS2063022  PMID: 16647080

Abstract

Simian immunodeficiency virus (SIV)-infection in macaques provides an important animal model for human immunodeficiency virus-1 (HIV-1) infection. The involvement of perforin (PFN), released by cytotoxic cells to mediate killing of virus-infected cells, has been difficult to assess in this experimental model due to a lack of reagents. We therefore evaluated monoclonal antibodies (mAbs) Pf-80, Pf-164 and Pf-344, previously raised against human PFN, for cross-reactivity with macaque PFN. Mabs Pf-164 and Pf-344 reacted with intracellular PFN in peripheral blood mononuclear cells (PBMC) from cynomolgus and rhesus macaques by flow cytometry and stained PFN in rhesus lymphoid tissue by immunohistochemistry (IHC). Moreover, PFN capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays utilizing mAbs Pf-164/Pf-80 for capture and mAb Pf-344 for detection were used to quantify PFN release by mitogen-stimulated cynomolgus and rhesus PBMC. The PFN ELISpot was further used to quantify antigen-specific CD8+ T cells by ex vivo stimulation of PBMC from cynomolgus macaques immunized against SIV/HIV-1. These macaque PFN-reactive mAbs and immunoassays will be valuable new tools for investigation of cytotoxic T lymphocyte (CTL) responses in non-human primate models of infectious diseases as well as for vaccine development.

Keywords: Perforin, Macaque, Monoclonal antibody, ELISpot, Capture ELISA, Flow cytometry, Immunohistochemistry

1. Introduction

The cynomolgus (Macaca fascicularis) and the rhesus macaque (Macaca mulatta) are commonly used in biomedical research in a wide variety of areas including basic and applied immunology, tumour biology, transplantation, virology, infectious diseases and vaccine development. Importantly, these macaques can be infected by simian immunodeficiency virus (SIV), which causes acquired immunodeficiency syndrome (AIDS)-like disease. Macaques therefore provide an important animal model for human immunodeficiency virus-1 (HIV-1) infection and are tools in the development of vaccines against HIV-1 (Haigwood, 2004). A major issue in this research area concerns the relevance of different in vitro-based methods used to quantify the degree of protective immunity elicited. A multitude of vaccines and immunization approaches have been evaluated, but still no clear in vitro correlate of protection has been defined (Pantaleo and Koup, 2004).

Perforin (PFN) is a protein released, from cytotoxic T lymphocytes (CTL) and natural killer cells (NK) cells, which mediates cytolysis of virus-infected cells. The cytolytic process involves degranulation of several proteins including PFN and granzymes, which together mediate target cell apoptosis (Russell and Ley, 2002). PFN is essential for the control of some viral infections, has a partial role in some, and appears to play a lesser role in others (Walsh et al., 1994; Kagi et al., 1995; Russell and Ley, 2002; Badovinac et al., 2003). The functional relevance of PFN in SIV/HIV-1 infection is an area of active investigation. HIV-1-specific CD8+ T cells in most patients harbor low levels of PFN (Appay et al., 2000; Champagne et al., 2001; Zhang et al., 2003), and may be impaired in cytolytic function (Appay et al., 2000; Lieberman et al., 2001). In addition, CD8+ T cells localized to lymphoid tissues, including lymph node (Andersson et al., 1999) and gastrointestinal mucosa (Shacklett et al., 2004) display limited PFN expression. In contrast, HIV-1-specific CD8+ T cells from HIV-1-positive long-term nonprogressors, but not progressors, strongly upregulate PFN levels when stimulated with HIV-1 infected autologous CD4+ T cells (Migueles et al., 2002). These findings point to an important role for PFN in immune control of HIV-1. However, the role of PFN in SIV infection has been difficult to assess due to a lack of appropriate reagents and methods for detection of intracellular and released macaque PFN.

Identification of mAbs to human PFN that display cross-reactivity with macaque PFN would provide the necessary means to analyze macaque PFN by various immunoassays. Due to the generally high degree of conservation of genes between humans and macaques, mAbs to human proteins frequently cross-react with the corresponding macaque protein although the degree of cross-reactivity varies and needs to be properly evaluated for each mAb (Makitalo et al., 2002). The homology between human and macaque PFN has not been defined but could be estimated to be around 95–100% at the amino acid level based on comparative data on cytokines (Villinger et al., 1995). Whereas single cross-reactive mAbs can be utilized for methods such as western blotting, flow cytometry and immunohistochemistry, capture ELISA and ELISpot assays require combinations of several mAbs that display a high level of cross-reactivity. Quantification of IFN-γ-producing antigen-specific CD8+ T cells by ELISpot is routinely used as a surrogate marker for CTL activity in acquired immunodeficiency syndrome (AIDS) vaccine research (Beattie et al., 2004; Makitalo et al., 2004; Cebere et al., 2005). The development of a PFN ELISpot for macaques could provide a complementary assay that identifies a response more functionally linked to CTL killing.

In the present study, we evaluated previously established human PFN-specific mAbs (Zuber et al., 2005) for their cross-reactivity with macaque PFN and their ability to detect macaque PFN by intracellular flow cytometry and immunohistochemical (IHC) tissue staining. Moreover, a combination of mAbs with different epitope specificities was used to quantitate macaque PFN by capture ELISA and to quantify the number of PFN-releasing cells by ELISpot. Using these new tools we were able to measure immune responses induced by a chimeric simian–human immunodeficiency virus (SHIV) vaccine.

2. Materials and methods

2.1. Monoclonal antibodies to human PFN

MAb Pf-80, Pf-164, Pf-344 (Mabtech, Nacka Strand, Sweden) and mAb δG9 (BD Pharmingen, Franklin Lakes, NJ, USA) were used in the study. MAbs were biotinylated and FITC labeled as described previously (Zuber et al., 2005).

2.2. Peripheral blood mononuclear cells (PBMC)

2.2.1. Cells used in flow cytometry

Venous blood from cynomolgus and rhesus macaques as well as from human volunteers was collected in EDTA-treated tubes (Becton-Dickinson, Franklin Lakes, NJ, USA). Animal handling and human blood sampling were in accordance with the ethical guidelines of the Animal Care and Use Committee, California National Primate Research Center, and the Institutional Review Board of the University of California, Davis. PBMC were isolated from the blood using Ficoll-Paque™ (Amersham Pharmacia Biotech, Uppsala, Sweden) centrifugation and the PBMC were cryopreserved. Prior to the experiments, PBMC were washed in phosphate buffered saline (PBS)/2% fetal calf serum (FCS).

2.2.2. PBMC used in ELISA and ELISpot

Housing and care procedures for cynomolgus macaques were in compliance with the provisions and general guidelines of the Swedish Animal Welfare Agency, and all procedures were approved by the Local Ethical Committee on Animal Experiments. PBMC were isolated, cryopreserved and thawed as described (Makitalo et al., 2002). CD8+ T cells were depleted from PBMC using Dynabeads (Dynal Biotech, Oslo, Norway) according to the manufacturer’s instructions.

2.3. Flow cytometry

Permeabilisation of macaque and human PBMC was performed using Fix and Perm reagents (Caltag Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Surface staining reagents recognized both human and macaque antigens and included anti-CD3-PerCP (clone SP34-2; BD Pharmingen, San Jose, CA, USA), and anti-CD8-APCCy7 (clone SK1; BDIS) mAbs. For intracellular staining, anti-human PFN-FITC mAbs were used. After staining, cells were fixed in 1% formaldehyde and analyzed on a FACScan (Becton-Dickinson, Franklin Lakes, NJ, USA) upgraded with ared laser (Cytek Development, Fremont, CA, USA). Spectral compensation and data analysis were done with FlowJo software (Tree Star, Inc., Ashland, OR, USA).

2.4. Immunohistochemistry

Upon harvest at necropsy, spleen tissue from normal rhesus macaques was embedded in Optimal Cutting Temperature (Sakura Finetek, Torrance, CA, USA) and snap frozen. Eight micrometer thick sections were cut on a cryostat, adhered to silinized glass slides, air-dried and fixed in 2% formaldehyde for 12 min. Slides were stored at −20 °C until staining. All solutions were made up in 0.1% saponin (Sigma, St. Louis, MO, USA) in Earle’s Balanced Salt Solution (Gibco, Grant Island, NY, USA), pH 7. Endogenous tissue peroxidase activity was prevented by treating the tissue sections in 1% H2O2 solution for 30 min. The sections were blocked with an avidin–biotin kit from Vector Laboratories (Burlingham, CA, USA) and with a 5% bovine serum albumin (BSA) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), 5% natural goat serum solution (Gibco) for 30 min before overnight incubation with the primary antibodies at room temperature (RT). MAbs Pf-344, Pf-164 and Pf-80 were used at a concentration of 0.5 μg/ml. Following gentle rinsing, biotinylated goat anti-mouse IgG (Vector Laboratories) was applied for 30 min followed by detection with an avidin–biotin horseradish peroxidase complex (Vectastain Elite, Vector Laboratories). 3′-diaminobenzidine tetrahydrochloride (DAB from Vector Laboratories) was used as a substrate to yield a deep brown color. Sections were counterstained with Mayer’s Haemotoxylin and mounted. Images were obtained on a Leica DMX-R microscope.

2.5. Capture ELISA

The ELISA assay was performed as described (Zuber et al., 2005). Briefly, EIA plates were adsorbed overnight at 4 °C with 100 μl of mAb Pf-80 or Pf-164 at 4 μg/ml or with 2 μg/ml of each mAb in PBS. For detection, 100 μl of biotinylated mAb Pf-344 at 1 μg/ml was used. The samples assessed were cell supernatants diluted 1/2 in PBS with 0.1% BSA, 0.15% Kathon CG and 0.05% Tween-20 (incubation buffer). The concentration of macaque PFN in the sample supernatants was determined by comparison to a human PFN standard (Mabtech).

2.6. ELISpot

PFN ELISpot was performed as described (Zuber et al., 2005). Briefly, PVDF membrane plates (Millipore, Bedford, MA, USA) were pre-wetted with EtOH, washed with sterile H2O and coated with 100 μlof mAbs Pf-80 and Pf-164, each at 15 μg/ml, in PBS overnight at 4 °C. The wells were washed with PBS and blocked with RPMI with 10% FCS. After that, 50 μl cell medium (RPMI-1640 supplemented with 10% FCS, 2 mM l-glutamine, 100 IU/ml Penicillin and Streptomycin and 1% HEPES, all from Life Technologies, Glasgow, UK) with or without stimuli and 50 μl of cells (200,000 per well) in cell medium were added per well and incubated in the plates overnight at 37 °C and 5% CO2. The next day, the cell suspension was discarded and the plates were washed with PBS. Biotinylated mAb Pf-344 at 1 μg/ml in PBS with 0.5% FCS was added and incubated at RT for 2 h. After washing with PBS, streptavidin-alkaline phosphatase (Mabtech) diluted 1/1000 in PBS with 0.5% FCS was added and incubated for 1 h at RT. After washing, filtrated substrate BCIP/NBT Plus (Mabtech) was added and incubated for 40 min. The reaction was stopped by rinsing the plates in tap water. The plates were dried and analyzed by using an automated AID EliSpot Reader System (AID, Strassberg, Germany). Macaque IFN-γ ELISpot was performed in parallel using mAb GZ-4 for capture (15 μg/ml) and mAb 7-B6-1-biotin (1 μg/ml) for detection (Mabtech).

2.7. Vaccine constructs and peptides

Cynomolgus macaques were immunized as previously described (Makitalo et al., 2004). Briefly the macaques were primed with DNA (0.5 mg/construct, totally 2.5 mg/immunization) and boosted with MVA (108 pfu/construct, totally 5×108 pfu/immunisation) both encoding SIVmac239 Gag/Pol and HIV-1IIIB Nef, Tat, Rev and gp120. Frozen PBMCs from three vaccinated macaques, selected on the basis of having an IFN-γ response against SIV Gag, were used to evaluate antigen-specific PFN responses. A pool of SIVmac251 Gag peptides (20-mer peptides with a 10 amino acid overlap; in total 49 peptides) (ARP 714.1-22, EVA 775.1-13 AND EVA 776.1-14; centralized facility for AIDS reagents, EU programme, EVA) at a concentration of 2.5 μg of each peptide/ml was used to stimulate macaque PBMCs in the ELISpot assay.

3. Results

3.1. Flow cytometry analysis of macaque PBMCs

The anti-human mAbs Pf-80, Pf-164, Pf-344 and mAb δG9 were analyzed for their reactivity with unstimulated human, cynomolgus and rhesus PBMCs by flow cytometry. The CD3+ lymphocyte population was gated and staining of the surface marker CD8 and intracellular staining of PFN analyzed. Positive staining of PFN was observed with mAbs Pf-344 and Pf-164 using human, cynomolgus and rhesus PBMCs, while Pf-80 and δG9 stained PFN in human but not macaque cells (Fig. 1). The PFN+ cells, in all three species, included CD8high, CD8low and CD8− cells.

Fig. 1.

Fig. 1.

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Detection of intracellular PFN by three color flow cytometry. Unstimulated rhesus, cynomolgus and human PBMC were stained for surface markers CD3 and CD8 as well as for intracellular PFN using four different human PFN-specific mAbs (Pf-80, Pf-164, Pf-344 and δG9). The plots depict CD8+ and PFN+ expression in the CD3+ lymphocyte population. Representative data are shown from multiple humans, cynomolgus macaques and rhesus macaques tested in at least four experiments.

3.2. PFN expression in macaque lymphoid tissue

The PFN-specific mAbs Pf-80, Pf-164 and Pf-344 were further evaluated for staining of lymphoid tissue inrhesus macaque spleen sections by IHC. The optimal staining in terms of frequency of positive cells and intensity of staining was obtained using mAbs Pf-344 and Pf-164 (Fig. 2) whereas Pf-80 was less efficient (data not shown). The staining pattern showed the granular and polarized expression of PFN (Fig. 2), in line with previously described stainings in human tissue (Andersson et al., 1999; Zuber et al., 2005).

Fig. 2.

Fig. 2.

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Immunohistochemical PFN staining of spleen from rhesus macaques. Tissue sections from a healthy rhesus macaque were stained using PFN-specific mAbs Pf-164 or Pf-344. The brown color indicates positive PFN staining while the cell nuclei were stained blue by haemotoxylin. All stainings were performed under identical experimental conditions.

3.3. mAb reactivity with macaque PFN, analyzed by capture ELISA

We next tested the ability of human PFN-specific mAb combinations to function as matched capture and detection mAb pairs in an ELISA assay for quantification of macaque PFN. Supernatants from PHA-stimulated cynomolgus and rhesus macaque PBMC were analyzed for PFN content. As shown in Fig. 3A, both mAb Pf-80 and Pf-164 were able to serve as capture antibodies in this context when mAb Pf-344-biotin was used as a detection antibody. Although Pf-80 could not be used to detect macaque PFN by flow cytometry, this antibody was found to cross-react with macaque PFN in the context of an ELISA (Fig. 3A). By combining the two capture mAbs, we were able to increase the signal intensity for detection of PFN in both macaque species. Quantification of the PFN content in supernatants from unstimulated and PHA-stimulated (48 h) PBMC from rhesus and cynomolgus macaques yielded concentrations of approximately 0.5 ng/ml of PFN after PHA stimulation whereas the supernatants from unstimulated cells were devoid of detectable PFN.

Fig. 3.

Fig. 3.

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ELISA analysis of cynomolgus and rhesus macaque PFN. A) Supernatants diluted 1/2 from unstimulated or PHA-stimulated PBMC (48 h) from cynomolgus (n=2) and rhesus (n=3) were analyzed by ELISA using Pf-80 (4 μg/ml), Pf-164 (4 μg/ml) or a combination of Pf-80 (2 μg/ml)+Pf-164 (2 μg/ml) as capture mAb; mAb Pf-344-biotin was used as detection mAb in all assays. The absorbance signal obtained with supernatant from stimulated (PHA) or unstimulated (medium) cells is shown. B) Mean values and SD of PFN in supernatants from cynomolgus (n=2) and rhesus (n=3) PBMC cultured with or without PHA. The levels of macaque PFN were determined by comparison against a standard curve obtained by serial dilution of human PFN at a known concentration.

3.4. Vaccine-induced immune responses analyzed by PFN ELISpot

The mAb combination found to be optimal for use in PFN ELISA (Pf-80/Pf-164 for capture and Pf-344 for detection; Fig. 3A), was also optimal for ELISpot analysis of PHA-stimulated cynomolgus macaque PBMC (data not shown). PHA induced strong responses in PBMC from several macaques and backgrounds from unstimulated cells were low (data not shown). The ELISpot system was thereafter applied for analysis of PBMC from SHIV-immunized cynomolgus macaques. A strong antigen-specific response to Gag-peptide pool manifested by PFN release was observed in 2 out of 3 immunized macaques. The induction of PFN release induced by Gag peptides was abolished by CD8+ T cell depletion, indicating that the PFN was released by CD8+ T cells (Fig. 4). All 3 animals displayed strong Gag-peptide poolspecific IFN-γ responses as assessed by a macaque IFN-γ-specific ELISpot assay (Fig. 4). Noteworthy, in two monkeys IFN-γ secretion was not significantly abolished by CD8+ T cell depletion suggesting the involvement of CD4+ T cells instead.

Fig. 4.

Fig. 4.

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ELISpot analysis of CD8+ T cell-mediated SIV Gag-specific PFN and IFN-γ release by cynomolgus PBMC. PBMC (200,000/well) from three SIV/HIV-1-immunized cynomolgus macaques (E15, E17 and E20) were incubated with medium or with a pool of overlapping SIV Gag peptides and the number of cells (i.e. spots) releasing PFN or IFN-γ was assessed by ELISpot. To define the involvement of CD8+ cells, PBMC depleted of CD8+ cells were analyzed in parallel. Stimulation with PHA served as a positive control (not shown).

4. Discussion

In this study we evaluated a panel of mAbs directed against human PFN for cross-reactivity with PFN from cynomolgus and rhesus macaques, frequently used as challenge models in AIDS vaccine studies. Macaques can be experimentally infected with SIV or SHIV, which currently represents the most widely utilized animal model to study lentivirus pathogenesis (Haigwood, 2004). It is believed that induction of virus-specific CD8+ T-cell responses through vaccination is essential in order to achieve efficient, protective immunity against HIV-1 in humans (McMichael and Hanke, 2003). However, the phenotypic and functional characteristics CD8+ T cells that should have to be protective are not yet fully defined (Appay et al., 2002a; Kaech et al., 2002; Nixon et al., 2003; Aandahl et al., 2004; Betts et al., 2005). The cytokine IFN-γ is frequently used as a readout in many vaccine studies (Letsch and Scheibenbogen, 2003). However, human memory/effector CD8+ T cells also produce other cytokines and chemokines, such as TNF-α, IL-2, and MIP-1β (Betts et al., 2004, 2005), and there is considerable functional heterogeneity in CD8+ T-cell responses (Sandberg et al., 2001). Accordingly, there is a need to develop new methods for measuring production and release of additional cytokines, chemokines, and cytolytic granule constituents in humans as well as in macaques. PFN is a marker of cytolytic CTLs and NK cells that is directly involved in target cell killing (Stinchcombe et al., 2004). Healthy PFN expression in HIV-1-specific CD8+ T cells has also been associated with long-term nonprogressive HIV-1 infection (Migueles et al., 2002). In the SIV macaque model, intracellular and released PFN has been difficult to measure due to a lack of macaque PFN-specific immunoassays.

MAbs Pf-164 and Pf-344, previously raised against human PFN, detected PFN from both macaque species in flow cytometry and did, as previously observed in humans (Appay et al., 2002a), include CD3+ PFN+ cells that were CD8high, CD8low or CD8−. The CD3+ PFN+ CD8− population may consist of CD4+ T cells or γδ T cells. Human CD4+ PFN+ cells have been observed by us (Jordan et al., 2006) and by others (Appay et al., 2002b; Norris et al., 2004) but the identity of the macaque CD3+ PFN+ CD8− cells observed in the current study has to be further elucidated. MAbs Pf-164 and Pf-344 also detected rhesus PFN in IHC, and the observed polarized and granular staining pattern was similar to what has been observed in human tissue (Zuber et al., 2005). Although mAb Pf-80 did not stain macaque cells in flow cytometry it yielded a moderate IHC staining of rhesus tissue.

PFN from both macaque species could also be detected by capture ELISA. The combination of mAb Pf-164 for coating and mAb Pf-344 for detection resulted in a functional detection system which, could be further enhanced using a combination of both mAb Pf-80 and mAb Pf-164 for coating. The contribution of mAb Pf-80 in the capture ELISA could be due to enhanced avidity effects.

In our previous study of the PFN content in human PBMC culture supernatants, following PHA stimulation, we found considerably higher PFN levels than those shown here in macaques (Zuber et al., 2005). Notably, the standard curve used for the quantification of the macaque PFN was obtained using a serial dilution of human PFN at known concentrations. As with other capture ELISAs for monkey proteins that utilize anti-human protein antibodies and human standards, the quantification of monkey protein concentrations depends on the degree of cross-reactivity displayed by the antibodies. The overall higher level of PFN measured after stimulation of human cells versus macaques cells could thus depend on multiple factors; I) The mAbs used in the assay may display a better reactivity with human than macaque PFN; II) The PHA-induced response is similar in human and macaques but the magnitude of the response to PHA, or the optimal PHA concentration for stimulation could differ.

The IFN-γ ELISpot assay is often used to quantify antigen-specific T-cell responses in order to establish an estimate of the magnitude of CTL activity. The assay is easy and robust to perform but the analysis is to some extent complicated by the fact that both CD4+ and CD8+ T cells can respond to an antigen by IFN-γ production. Although this potential difficulty can be circumvented by various strategies, PFN ELISpot may facilitate a more direct assessment of CD8+ T-cell responses. This is supported by our finding that antigen-specific PFN release in PBMC from macaques immunized against SIV-HIV-1 antigens was completely abolished by depletion of CD8+ T cells. With regard to antigen-specific IFN-γ responses, the effect of depleting CD8+ T cells differed between monkeys suggesting a response involving CD4+ and/or CD8+ T cells.

In conclusion, the establishment of functional macaque PFN-reactive mAbs and immunoassays enables measurement of intracellular and released macaque PFN and will facilitate an improved understanding of the role of PFN in infectious disease and vaccine development.

Acknowledgements

This work was supported by the Swedish Foundation for Strategic Research, the Swedish International Development Agency, the Swedish Science Council, Karolinska Institutet, U.S.A. Public Health Service Grant V51-00169, NIH grants R01-A1-51239, R01-AI-48484 and R01-AI-57020. We acknowledge the technical expertise of Katy Lantz and Tracey Rourke.

Abbreviations:

CTL

cytotoxic T lymphocyte

ELISA

enzyme-linked immunosorbent assay

ELISpot

enzyme-linked immunospot assay

FCS

fetal calf serum

HIV-1

human immunodeficiency virus-1

IHC

immunohistochemistry

mAb

monoclonal antibody

PBMC

peripheral blood mononuclear cells

PBS

phosphate buffered saline

PFN

perforin

PHA

phytohemagglutinin

SIV

simian immunodeficiency virus

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