A Method for Detecting Intracellular Perforin in Mouse Lymphocytes

J Immunol 2014; 193:5744-5750; Prepublished online 27 October 2014; doi: 10.4049/jimmunol.1402207 http://www.jimmunol.org/content/193/11/5744

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 9650 Rockville Pike, Bethesda, MD 20814-3994. Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.

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Amelia J. Brennan, Imran G. House, Jane Oliaro, Kelly M. Ramsbottom, Magdalena Hagn, Hideo Yagita, Joseph A. Trapani and Ilia Voskoboinik

The Journal of Immunology

A Method for Detecting Intracellular Perforin in Mouse Lymphocytes Amelia J. Brennan,* Imran G. House,*,† Jane Oliaro,*,† Kelly M. Ramsbottom,* Magdalena Hagn,* Hideo Yagita,‡ Joseph A. Trapani,*,†,x and Ilia Voskoboinik*,†,{,‖

O

ur immune system is regulated by intricate and complex signaling pathways, which allow specific subsets of cells to communicate and ultimately protect us from disease. Cytotoxic lymphocytes (CLs) are specialized immune cells that recognize and destroy transformed cells and those carrying an intracellular pathogen. Whereas NK cells provide the initial rapid “innate” defense, cytotoxic T lymphocytes (CTLs) need to undergo a program of differentiation and represent the long-lived “adaptive” arm of the immune response. Nevertheless, both CTLs and NK cells destroy target cells primarily through a common, perforin-dependent mechanism (1–3). The formation of an immune synapse between a CL and target cell results in the polarization of specialized secretory granules toward the cell interface (4, 5). These granules are predicted to

*Cancer Immunology Program, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia; †Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria 3010, Australia; ‡Department of Immunology, Juntendo University School of Medicine, Tokyo 113-8421, Japan; x Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia; {Department of Pathology, University of Melbourne, Parkville, Victoria 3010, Australia; and ‖Department of Genetics, University of Melbourne, Parkville, Victoria 3010, Australia Received for publication August 27, 2014. Accepted for publication September 23, 2014. This work was supported by project and program grants from the National Health and Medical Research Council of Australia and the Cancer Council Victoria (to I.V. and J.A.T.). I.V. is supported by a National Health and Medical Research Council of Australia fellowship. I.G.H. is supported by an Australian postgraduate award. A.J.B. designed and conducted the study and cowrote the manuscript; I.G.H., J.O., M.H., and K.M.R. designed and conducted experiments; H.Y. produced and provided critical reagents; J.A.T. provided input on study design and cowrote the manuscript; and I.V. designed the study and cowrote the manuscript. Address correspondence and reprint requests to Dr. Ilia Voskoboinik and Dr. Amelia J. Brennan, Cancer Immunology Program, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, VIC 3002, Australia. E-mail addresses: ilia. [email protected] (I.V.) and [email protected] (A.J.B.) The online version of this article contains supplemental material. Abbreviations used in this article: CL, cytotoxic lymphocyte; CTL, cytotoxic T lymphocyte; PFA, paraformaldehyde. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402207

contain the pore-forming protein, perforin, and proapoptotic serine proteases, granzymes (6). Following granule fusion with the plasma membrane, granzymes and perforin monomers are released into the synaptic cleft, where perforin monomers oligomerize and rapidly form a transmembrane pore, through which granzymes diffuse into the cytoplasm of the target cell and initiate apoptosis (7, 8). Perforin is, therefore, the one essential component of the CL death machinery. Significant disruption in its delivery to the target cell leads to life-threatening immune-mediated disease and a predisposition to immunogenic malignancies, highlighting the critical role of perforin in immune surveillance of cancer and infections (9–12). However, the inability to detect perforin in mouse CTLs has, for decades, significantly impacted the study of many human diseases using experimental animal models (13, 14). In the present study, we report a novel technique that provides the most reliable and sensitive detection to date of intracellular mouse CTL perforin by confocal microscopy and flow cytometry. We have successfully used this method to investigate a number of important immunological questions related to perforin protein expression in mouse CTLs.

Materials and Methods Cell isolation, maintenance, and stimulation Primary CTLs of C57BL/6 mice that also express a transgenic TCR (OT-I), that is, OT-I Prf1+/+, OT-I Prf12/2, and OT-I Prf1+/2, were generated (15), activated, and maintained as previously described (16). Spleens were harvested from naive OT-I Prf1+/+, OT-I Prf12/2, and OT-I Prf1+/2 mice and pressed through a sterile 70-mm nylon mesh to generate single-cell suspensions. Naive mouse CTLs were isolated from spleens by negative selection using the MACS CTL isolation kit (Miltenyi Biotec). Purity of naive CTLs was verified by surface staining with CD8a and CD62L and analyzed by flow cytometry. For in vitro CTL activation, splenocytes were cultured with 10 nM OVA257 peptide and 100 U/ml recombinant human IL-2 for 3 d in supplemented RPMI 1640 media at 37˚C, 5% CO2 as previously described (15, 16). Purity and activation states of CTLs were verified by surface staining with CD8a, CD44, and CD62L and analyzed by flow cytometry. The mouse fibroblast cell line MC57 was maintained as previously described (17). All animal studies were performed according to the ethical guidelines approved by the Peter MacCallum Cancer Centre.

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Cytotoxic lymphocytes destroy pathogen-infected and transformed cells through the cytotoxic granule exocytosis death pathway, which is dependent on the delivery of proapoptotic granzymes into the target cell cytosol by the pore-forming protein, perforin. Despite the importance of mouse models in understanding the role of cytotoxic lymphocytes in immune-mediated disease and their role in cancer immune surveillance, no reliable intracellular detection method exists for mouse perforin. Consequently, rapid, flowbased assessment of cytotoxic potential has been problematic, and complex assays of function are generally required. In this study, we have developed a novel method for detecting perforin in primary mouse cytotoxic T lymphocytes by immunofluorescence and flow cytometry. We used this new technique to validate perforin colocalization with granzyme B in cytotoxic granules polarized to the immunological synapse, and to assess the expression of perforin in cytotoxic T lymphocytes at various stages of activation. The sensitivity of this technique also allowed us to distinguish perforin levels in Prf1+/+ and Prf1+/2 mice. This new methodology will have broad applications and contribute to advances within the fields of lymphocyte biology, infectious disease, and cancer. The Journal of Immunology, 2014, 193: 5744–5750.

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FIGURE 1. Intracellular perforin can be visualized in mouse CTLs by confocal microscopy after fixation with Bouin’s solution. (A) Confocal microscopy of OT-I Prf1+/+ CTLs 6 d after activation showed colocalization of perforin with other lymphocyte secretory granule proteins, granzyme B and LAMP-1. Left panel, For clarity, stains are shown in grayscale. Right panel, For clarity in the differential interference contrast image, stains are shown as: perforin, red; granzyme B, cyan; and DAPI, blue. For clarity in the merged imaged, stains are shown as: perforin, red; granzyme B, cyan; and LAMP-1, blue. (B) Confocal microscopy of immune synapses formed between OT-I CTLs 6 d after activation and MC57 target cells showed polarization of perforin and g-tubulin toward the synaptic cleft, but no colocalization. Left panel, For clarity, stains are shown in grayscale. Right panel, For clarity in the merged images, stains are shown as: perforin, red; g-tubulin, green; DAPI, blue. (C) Confocal microscopy of immune synapses formed between OT-I Prf1+/+ CTLs 6 d after activation and MC57 target cells show perforin and granzyme B colocalization in secretory granules polarized toward the synaptic cleft. Left panel, For clarity, stains are shown in grayscale. Right panel, For clarity in the merged images, stains are shown as: perforin, red; granzyme B, cyan; and DAPI, blue. Note that all confocal images are z-stacks and are representative of three independent experiments. Scale bars, 10 mm.

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FIGURE 2. Temporal expression of intracellular perforin in mouse CTLs was detected by flow cytometry after fixation with Bouin’s solution, but not with PFA. (A) Flow cytometry of naive OT-I Prf1+/+, OT-I Prf12/2, and OT-I Prf1+/2 CTLs (day 0) after fixation with Bouin’s solution and incubation of primary Abs for 16 h do not express intracellular perforin. Histograms are representative of two independent experiments. (B) Flow cytometry after fixation with Bouin’s solution and incubation of primary Abs for 16 h. Left panel, Temporal expression of intracellular perforin was detected by flow cytometry in OT-I Prf1+/+ CTLs but not in OT-I Prf12/2 CTLs. Right panels, Temporal expression of intracellular granzyme B and perforin was detected by flow cytometry in OT-I Prf1+/+ CTLs. FACS plots are representative of at least three independent experiments, using CTLs activated from a naive state. (C) Flow cytometry after fixation with PFA and incubation of primary Abs for 16 h. Left panel, Only background levels of intracellular perforin were detected by flow cytometry in OT-I Prf1+/+ CTLs over time. Right panels, Intracellular granzyme B, but only background levels of intracellular perforin, was detected by flow cytometry in OT-I Prf1+/+ CTLs over time. FACS plots are representative of at least three independent experiments, using CTLs activated from a naive state. (C) The temporal increase of intracellular perforin expression in OT-I CTLs activated from a naive state could be detected after fixation with Bouin’s solution but not with PFA. Perforin protein levels of OT-I Prf1+/+ CTLs were calculated as fold increase of (Figure legend continues)

The Journal of Immunology Abs

Intracellular staining for confocal microscopy OT-I Prf1+/+ and OT-I Prf12/2 CTLs were isolated and activated as described above. Six days after activation, CTLs were washed and resuspended in PBS at a concentration of 2 3 106 cells/ml. At room temperature, 2 3 105 cells were gently pipetted on a poly-L-lysine–coated microscope slide within a small boundary marked with a PAP pen (Invitrogen) to prevent runoff. The CTL suspension was left for 30 min to allow cells to settle on the slide as previously described (16). Once settled, PBS was gently removed and cells were fixed with paraformaldehyde (PFA) or Bouin’s solution (acetic acid 5%, formaldehyde 9%, picric acid 0.9%). For fixation with PFA, cells were fixed with 3.7% PFA for 15 min, followed by 33 1% BSA/13 PBS washes. Cells were then permeabilized with 0.1% Triton X-100/13 PBS for 10 min, followed by 33 0.1% BSA/13 PBS washes and blocking with 0.1% BSA/13 PBS for 1 h at 4˚C. For fixation with Bouin’s solution, cells were fixed with Bouin’s solution at room temperature for 20 min, followed by 33 1% BSA/13 PBS washes to remove all traces of picric acid and prevent autofluorescence. Cells were then permeabilized with 0.1% Triton X-100/13 PBS for 10 min, followed by 33 1% BSA/13 PBS washes and blocking with 0.1% BSA/13 PBS for 1 h at 4˚C. After fixation and permeabilization, primary Abs were diluted in 0.1% BSA/13 PBS and added to fixed CTLs for incubation at 4˚C for 1 or 16 h. The primary Abs were then removed with 33 0.1% BSA/13 PBS washes and secondary Abs were added for incubation at 4˚C for 1 h. The cells were imaged with an Olympus FV1000 confocal microscope equipped with a 405-nm UV laser, 12.9-mW 488-nm multi-ion argon laser, a 1-mW 543-nm green HeNe laser, and a 11-mW 633-nm red HeNe laser. All images were captured with a PlanApoN 360 oil immersion objective (numerical aperture of 1.42). The images were subsequently processed with the Olympus Micro FV10-ASW program. Final images are displayed as z-stack projections, and orthogonal projections were obtained with the Olympus Micro FV10-ASW program (sections, 0.2–0.4 nm).

Generation of immune cell conjugates MC57 cells were plated into eight-well chamber slides (Nalge Nunc) at a density of 1 3 105 cells overnight and incubated with 1 mM OVA257 peptide for 1 h the next day. Media were removed and 1 3 106 CTLs from OT-I Prf1+/+ and OT-I Prf12/2 6 d after activation were added to chamber slides for 45 min to allow conjugates to form. Cells were fixed and permeabilized with Bouin’s solution and Triton X-100, respectively, as described above.

Intracellular staining for flow cytometry OT-I Prf1+/+, OT-I Prf12/2, and OT-I Prf1+/2 CTLs were isolated and activated as described above. Cells were washed twice with 13 PBS and fixed with PFA or Bouin’s solution. For fixation with PFA, cells were fixed with BD Cytofix/Cytoperm (4% PFA; BD Biosciences) for 20 min on ice and washed twice with 13 BD Cytoperm/Cytowash (BD Biosciences). For fixation with Bouin’s solution, cells were washed twice with 13 PBS and fixed with 1 ml Bouin’s solution at room temperature for 20 min, followed

by 33 0.2% BSA/13 PBS washes to remove all traces of picric acid and prevent autofluorescence. Cells were then permeabilized at room temperature for 10 min with 0.1% Triton X-100/13 PBS, followed by 33 0.2% BSA/13 PBS washes. After fixation and permeabilization, primary Abs were diluted in 0.2% BSA/13 PBS and added to 1 3 105 fixed cells for incubation at 4˚C for 30 min or 16 h. The primary Abs were then removed with 33 0.2% BSA/13 PBS washes, and secondary Abs at the concentrations specified above were added for incubation at 4˚C for 30 min. Cells were then analyzed for surface marker expression or for intracellular perforin and granzyme B expression by flow cytometry with FlowJo (v8.8.6).

Quantitative real-time RT-PCR Mouse perforin RNA was collected from OT-I Prf1+/+ and OT-I Prf1+/2 using TRIzol (Ambion) extraction. cDNA was produced using Moloney murine leukemia virus reverse transcriptase (Promega) as per the manufacturer’s instructions. Quantitative real-time RT-PCR was performed using a Rotor-Gene Q (Qiagen) and tracked using the intercalating fluorescent dye SYTO9 (Invitrogen). Prf1 expression was assessed using the primers 59-TCATCATCCCAGCCGTAGT-39 and 59-ATTCATGCCAGTGTGAGTGC-39, and the control gene HPRT was assed using the primers 59CTCTCGAAGTGTTGGATACAG-39 and 59-GACACAAACGTGATTCAAATCC-39. Prf1 expression for each sample was standardized by calculating the relative expression to control gene HPRT (22DDCt).

Results Visualization of intracellular perforin in mouse CTLs by confocal microscopy To date, there is no reliable technique for detecting intracellular mouse CTL perforin by confocal microscopy. In an attempt to resolve this issue, we fixed activated OT-I Prf1+/+ CTLs with Bouin’s solution (see Materials and Methods). Bouin’s solution is most commonly used for immunohistochemistry, and occasionally in confocal microscopy, due to its excellent preservation of cellular morphology and intracellular organelles (19, 20). Incubation of fixed and permeabilized CTLs with primary antiperforin Ab for 1 h at 4˚C did not result in any detectable perforin protein immunofluorescence. However, extending incubation to 16 h at 4˚C resulted in a clear perforin signal in OT-I Prf1+/+ CTLs (Fig. 1A). Perforin colocalized with another lymphocyte effector protein, granzyme B, and an abundant secretory granule protein, LAMP1. Not all CTLs expressed detectable amounts of perforin, and the levels of perforin and granzyme B within different secretory granules of the same cell were also heterogeneous (Fig. 1A). A threedimensional reconstruction of this image showed that intracellular morphology was well maintained, with clear positioning of individual and clustered secretory granules (Supplemental Fig. 1, Supplemental Video 1). We were unable to detect perforin in CTLs fixed with PFA. Next, we investigated the subcellular localization of perforin during immune synapse formation between OT-I Prf1+/+ CTLs and target cells (OVA257 peptide-pulsed MC57s). We confirmed the formation of the immune synapse by detecting recruitment of a marker of the microtubule organizing center, g-tubulin, to the interface between the CTL and a target cell. Importantly, perforin was also recruited to the interface between the two cells, but it did not colocalize with g-tubulin (Fig. 1B). Additionally, we demonstrated that both perforin and granzyme B colocalized at the immune synapse (Fig. 1C). Detection of intracellular mouse perforin in CTLs by flow cytometry The inability to detect intracellular mouse perforin by flow cytometry has been a significant obstacle in cellular immunology. We have now optimized perforin detection by flow cytometry using

geometric mean fluorescence intensity above isotype controls, and subtracted from corresponding OT-I Prf12/2 CTLs. All data points are the average of at least three independent experiments.

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For confocal microscopy, Abs used were rat monoclonal anti-mouse perforin [P1-8 (18), provided by Kyowa Kirin], mouse monoclonal antihuman granzyme B conjugated to allophycocyanin (BD Biosciences), rabbit polyclonal anti-mouse LAMP1 (Sapphire Bioscience), and mouse monoclonal anti-mouse g-tubulin (immune synapse marker, SigmaAldrich), and DAPI was used as a nuclear stain (Molecular Probes). Alexa Fluor 488–conjugated goat anti-rat Ab and Alexa Fluor 546–conjugated goat anti-mouse Ab were used to detect primary Abs. For flow cytometry, CTL surface marker Abs used were rat anti-mouse CD8a, rat anti-mouse Va2, rat anti-mouse CD62L, rat anti-mouse CD44, rat anti-mouse CD69, and hamster anti-mouse TCRb (all from BD Biosciences). For intracellular staining, rat monoclonal anti-mouse perforin [1 ng/ml, P1-8 (18)] and mouse monoclonal anti-human granzyme B conjugated to allophycocyanin (1 ng/ml, BD Biosciences) were used. Isotype-matched control rat IgG2a (eBioscience) and mouse IgG1 directly conjugated to allophycocyanin (R&D Systems) were used as negative controls. PE-conjugated goat anti-rat IgG Ab (1:200, Invitrogen) was used to detect primary Abs.

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Dynamic range and detection sensitivity of mouse perforin in lymphocytes The sensitivity and dynamic range of our new method enabled perforin levels in CTLs expressing a single functional Prf1 allele (OT-I Prf1+/2) to be easily distinguished from OT-I Prf1+/+ CTLs (Fig. 3A). Once again, Bouin’s solution was superior to PFA fixation, which provided no discernable perforin signal in OT-I Prf1+/2 compared with isotype controls (Fig. 3A). The difference in protein expression between cell types was not due to differential activation of the cells, which was indistinguishable, based on CD44 and CD62L levels (Supplemental Fig. 3). Previous studies have shown that upregulation of perforin protein in mouse NK cells is dependent on translation of a constitutively transcribed pool of perforin mRNA following cytokine stimulation (21). Owing to the lack of a reliable detection method, however, it remained unclear whether perforin protein expression levels are regulated by the same mechanism during CTL differentiation from a naive state. We found that intracellular perforin expression in OT-I Prf1+/+ CTLs gradually increased over time and peaked by day 10 after activation. However, unlike in NK cells, mRNA and protein expression showed similar kinetics (Fig. 3B). Investigation of OT-I Prf1+/2 also demonstrated similar kinetics, but mRNA and protein expression were reduced by ∼50% compared with the levels observed in OT-I Prf1+/+ CTLs (Fig. 3B).

Discussion

FIGURE 3. Detection of perforin protein in OT-I Prf1+/2 CTLs. (A) Intracellular perforin was detected by flow cytometry in OT-I Prf1+/+ CTLs and at a reduced level in OT-I Prf1+/2 after fixation in Bouin’s solution and incubation with primary Ab for 16 h. In comparison, no intracellular perforin was detected by flow cytometry in OT-I Prf1+/2 CTLs after fixation in PFA and incubation with primary Ab for 16 h. Histograms are representative of three independent experiments. (B) Kinetics of the perforin protein and gene expression in CTLs activated from a naive state. The relative perforin protein expression (left y-axis), as well as the corresponding relative perforin mRNA expression (right y-axis), in OT-I Prf1+/+ and OT-I Prf1+/2 CTLs after fixation with Bouin’s solution was calculated for 5–10 d after activation. Perforin protein levels were calculated as fold

Experimental mouse models are an invaluable scientific resource that allow comprehensive investigation of important biological questions in vivo and provide an essential platform to study many human diseases. However, the absence of a reliable detection method for the essential effector molecule, perforin, in mouse lymphocytes has hindered many studies. Our novel method provides reliable and quantitative techniques for detecting intracellular mouse perforin in these cells by both confocal microscopy and flow cytometry. We validated the dynamic range of this technique to show that CTLs expressing a single functional Prf1 allele can be easily distinguished from those of wild-type mice. We then applied the methodology to demonstrate that the kinetics of perforin expres-

increase of geometric mean fluorescence intensity above the isotype controls, and subtracted from corresponding OT-I Prf12/2 CTLs. The relative perforin mRNA expression was calculated as fold increase as standardized against day 5. All data points are the average of at least three independent experiments.

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Bouin’s solution. As expected, no perforin expression was present in naive CTLs (Fig. 2A). However, a significant perforin signal was detected in activated OT-I Prf1+/+ CTLs, following labeling with primary anti-perforin Ab for 16 h at 4˚C (Fig. 2B, Bouin’s solution, and summarized in Fig. 2C; no signal was detected after 30 min [data not shown]). In stark contrast, we found fixation with PFA was unreliable, as only low and inconsistent levels of perforin expression were detected (Fig. 2B, PFA fixative, and summarized in Fig. 2C). Similar to our confocal microscopy results, not all CTLs expressed detectable levels of perforin. Background staining was constantly low in OT-I Prf12/2 CTLs for up to 9 d after in vitro stimulation but became appreciable by day 10. Caution should therefore be applied to interpreting results with very long-term CTL stimulation. Importantly, fixation with Bouin’s solution permitted detection of other immune proteins, including intracellular granzyme B (Fig. 2B) and also cell surface markers such as CD8a, CD44, CD62L, CD69, Va2, and TCRb (Supplemental Fig. 2).

The Journal of Immunology

Acknowledgments We thank Drs. Daniel Andrews and Misty Jenkins for helpful suggestions and critical reading of the manuscript.

Disclosures The authors have no financial conflicts of interest.

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Cell 111: 837–851. ¨ ijo¨, J. Bassein, R. Chen, 27. Trifari, S., M. E. Pipkin, H. S. Bandukwala, T. A G. J. Martinez, and A. Rao. 2013. MicroRNA-directed program of cytotoxic + CD8 T-cell differentiation. Proc. Natl. Acad. Sci. USA 110: 18608–18613. 28. Kim, N., M. Kim, S. Yun, J. Doh, P. D. Greenberg, T. D. Kim, and I. Choi. 2014. MicroRNA-150 regulates the cytotoxicity of natural killers by targeting perforin1. J. Allergy Clin. Immunol. 134: 195–203. 29. Johnson, B. J., E. O. Costelloe, D. R. Fitzpatrick, J. B. Haanen, T. N. Schumacher, L. E. Brown, and A. Kelso. 2003. Single-cell perforin and granzyme expression reveals the anatomical localization of effector CD8+ T cells in influenza virus-infected mice. Proc. Natl. Acad. Sci. USA 100: 2657–2662. 30. Zaiss, D. M., A. J. Sijts, and T. R. Mosmann. 2008. Enumeration of cytotoxic CD8 T cells ex vivo during the response to Listeria monocytogenes infection. Infect. Immun. 76: 4609–4614.

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sion in activated naive OT-I Prf1+/+ CTLs correlates with ongoing Prf1 transcription. These data are consistent with previous reports, which show that once naive CTLs are activated through the TCR, an additional signal from IL-2 induces perforin mRNA transcription (22, 23) and acquisition of cytotoxic function (24). In the present study, we formally demonstrated that this is also associated with increased perforin protein levels. This is in stark contrast to mouse NK cells, which constitutively express a pool of perforin mRNA while exhibiting minimal protein expression and cytotoxicity prior to further stimulation with the cytokines IL-2 and IL-15 (21). These results reflect the differences observed in mouse CL perforin regulation and cytotoxicity: NK cells engage and kill targets via the perforin pathway within hours of encountering foreign Ag (21, 25), whereas naive CTLs express the highest level of perforin mRNA and protein ∼1 wk after TCR stimulation (22, 26). Interestingly, recent studies have highlighted a role for microRNA in the regulation of perforin expression in both mouse CTLs and NK cells (27, 28). The development of a detection method for intracellular perforin, therefore, provides a useful tool for further characterization of posttranscriptional regulation in mouse CLs. Consistent with previous reports, we found that not all mouse CTLs activated from a naive state express perforin (29–31). Similar to humans, the present study and others have shown that an increase in perforin expression is associated with differentiation of CTLs from a central memory to an effector memory phenotype (32, 33), which correlates with cytotoxic activity (34). However, formal characterization of perforin-expressing and cytotoxic mouse CTL subsets has not been possible in the past. Furthermore, because perforin is the one essential component of the CTL death machinery, identification of whether cytotoxic effector cells have successfully infiltrated the tumor microenvironment in experimental cancer models has been very difficult to assess. Current detection methods involve complicated cytotoxic assays using CTLs isolated from tumor tissue, which require in vitro enrichment and subsequent sorting by flow cytometry or negative selection. The detection of granzyme B+ CTLs is also used as a marker for cytotoxic tumor infiltrates; however, as shown in the present study and in the past, given that granzyme B is upregulated earlier than perforin (22, 34, 35), and not all granzyme B–expressing cells coexpress perforin (31), this technique is not necessarily a predictor of cytotoxicity. The development of a novel perforin detection method, therefore, provides a complementary and accurate means of addressing these critical immunological questions, particularly in experimental mouse models of human disease. Additionally, the dynamic range of this technique will also enable investigation of perforin expression and cytotoxic phenotype in other murine lymphocytes, such as gd T cells (36), NKT cells (37), and regulatory T cells.

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31. Peixoto, A., C. Evaristo, I. Munitic, M. Monteiro, A. Charbit, B. Rocha, and H. Veiga-Fernandes. 2007. CD8 single-cell gene coexpression reveals three different effector types present at distinct phases of the immune response. J. Exp. Med. 204: 1193–1205. 32. Hamann, D., P. A. Baars, M. H. Rep, B. Hooibrink, S. R. Kerkhof-Garde, M. R. Klein, and R. A. van Lier. 1997. Phenotypic and functional separation of memory and effector human CD8+ T cells. J. Exp. Med. 186: 1407–1418. 33. Kelso, A., E. O. Costelloe, B. J. Johnson, P. Groves, K. Buttigieg, and D. R. Fitzpatrick. 2002. The genes for perforin, granzymes A-C and IFN-gamma are differentially expressed in single CD8+ T cells during primary activation. Int. Immunol. 14: 605–613. 34. Jenkins, M. R., J. Mintern, N. L. La Gruta, K. Kedzierska, P. C. Doherty, and S. J. Turner. 2008. Cell cycle-related acquisition of cytotoxic mediators defines

the progressive differentiation to effector status for virus-specific CD8+ T cells. J. Immunol. 181: 3818–3822. 35. Jenkins, M. R., K. Kedzierska, P. C. Doherty, and S. J. Turner. 2007. Heterogeneity of effector phenotype for acute phase and memory influenza A virusspecific CTL. J. Immunol. 179: 64–70. 36. Wakita, D., K. Sumida, Y. Iwakura, H. Nishikawa, T. Ohkuri, K. Chamoto, H. Kitamura, and T. Nishimura. 2010. Tumor-infiltrating IL-17-producing gd T cells support the progression of tumor by promoting angiogenesis. Eur. J. Immunol. 40: 1927–1937. 37. Gumperz, J. E., S. Miyake, T. Yamamura, and M. B. Brenner. 2002. Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J. Exp. Med. 195: 625–636.

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A method for detecting intracellular perforin in mouse lymphocytes.

Cytotoxic lymphocytes destroy pathogen-infected and transformed cells through the cytotoxic granule exocytosis death pathway, which is dependent on th...
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