This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-γ (IFN-γ) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.
L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-γ by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-γ production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-γ production have an impact on IFN-γ responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-γ production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-γ responses to L. monocytogenes and survival of mice from this infection.
IFN-γ is a cytokine that is crucial for mediating immunity against intracellular pathogens and for controlling tumor growth5. The importance of this cytokine in bacterial resistance is evident in the observation that humans with mutations in the IFN-γ signaling pathway are highly susceptible to infection with mycobacteria and salmonellae6. Similarly, mice deficient in either IFN-γ or the IFN-γ receptor exhibit defects in resistance to mycobacteria7-9 and other intracellular pathogens including L. monocytogenes10,11, Leishmania major12, Salmonella typhimurium13, and certain viruses11. In addition to combatting pathogens, IFN-γ plays a crucial role in host-defense against tumors14. Though higher production of IFN-γ is beneficial in the context of infection or cancer, prolonged production of this cytokine has been linked to the development of systemic autoimmunity15-17 and the acceleration of type I diabetes in the non-obese diabetic mouse model18.
The major sources of IFN-γ include NK cells, NKT cells, γδ T cells, T helper 1 (Th1) cells, and cytotoxic T lymphocytes (CTL)5,19,20. IFN-γ enhances both innate and adaptive immunity by: (1) up-regulating major histocompatibility complex (MHC) class I and II expression, (2) increasing the expression of co-stimulatory molecules on antigen presenting cells, (3) enhancing macrophage phagocytosis and the production of pro-inflammatory cytokines and microbicidal factors (e.g., nitric oxide and reactive oxygen species), (4) promoting the differentiation of naïve CD4+ T cells into Th1 effector cells, (5) promoting antibody class switching to immunoglobulin (Ig)2a and IgG3 (in mouse), (6) inducing the production of chemokines to recruit immune cells to sites of infection, and (7) enhancing NK cell and CTL responses5,19. Given the crucial importance of IFN-γ in the host response to pathogens and tumors, recombinant IFN-γ has been tested as a treatment for various infections and malignancies (reviewed in19). However, because systemic administration of IFN-γ or the Th1 promoting cytokine interleukin-12 (IL-12) is associated with side effects and dose-related toxicity19,21, there is interest in developing alternative strategies to increase IFN-γ production by immune cells. Development of new biologics and small molecules requires in vivo screening tools to test whether such agents increase IFN-γ production during an immune response and whether this translates into meaningful biological effects such as increases in animal survival.
Experimental infection of mice with the gram-positive bacterium L. monocytogenes has been an instrumental model for deciphering the role of IFN-γ in host-immunity against intracellular pathogens1,22. Infection of mice with the pathogen intravenously or intraperitoneally (i.p.) leads to the rapid dissemination of the bacteria to the spleen and liver, where they become internalized by resident macrophages and hepatocytes with peak bacterial loads in the spleen occurring between 3 and 4 days post-infection1,3,22. Production of IFN-γ by NK cells is important for macrophage activation and early resistance against the pathogen3; however at high infectious doses, production of IFN-γ can also be detrimental to pathogen clearance23. NKT cells are also a source of IFN-γ in the spleen and liver during early control of pathogens2,24 and this production has been shown to amplify IFN-γ production by other cell types including NK cells2. On the other hand, later-acting adaptive T lymphocytes, CD8+ T cells in particular, are important for mediating the clearance of the pathogen and providing protection against re-infection1,4,22.
This infection model has been attractive to researchers for a number of reasons (reviewed in1). First, infection with the pathogen is highly reproducible and induces a strong Th1 and cellular immune response. Secondly, during sublethal infection, bacterial load is concentrated in the liver and spleen where it can be easily measured. Thirdly, the pathogen can be safely handled under Biosafety Level 2 (BSL2) conditions. Fourthly, the organism and the immune response that it generates have been extensively characterized. Finally, a variety of mutant and genetically-modified strains have been developed that are available for use.
Described here is a basic protocol for inoculation of C57BL/6J mice with the EGD strain of L. monocytogenes25 and for measuring IFN–γ responses by NK, NKT, and adaptive lymphocytes post-infection. Also described is how to measure bacterial load in the spleen and liver after sublethal infection and to carry out survival studies after infection with a modified LD50 dose of the pathogen. Finally, representative data are shown of how this protocol can be used to screen the effect of new treatments on IFN-γ responses and mouse survival from L. monocytogenes infection.
Safety Statement
This protocol describes infection of mice with live L. monocytogenes. The pathogen is handled safely under BSL2 conditions by trained personnel who are not immunocompromised. Immunocompromised people include pregnant women, the elderly and individuals who are HIV-infected or have chronic conditions that require treatment with immunosuppressive therapy. Personnel should don a protective lab coat or gown, gloves, mask, and eye protection while handling infected samples. The work described herein was performed under BSL2 conditions under a certificate (#32876) that was issued by the University Health Network (UHN) Biosafety office. Carcasses from infected mice or any unused tissues were double-bagged and disposed of in biohazard waste. Cages from infected mice were also decontaminated by autoclaving.
Ethics Statement
Mice were maintained and infected in a quarantine room within UHN animal facilities and were cared for in accordance with the guidelines set by the Canadian Council on Animal Care. All procedures on mice were carried out under animal use protocol #3214 that was approved by the UHN animal care committee. Due to ethical considerations, death was not used as an endpoint for survival studies. The modified LD50 dose reported here for L. monocytogenes infection was determined to be the dose at which 50% of the mice reached specific endpoints, which consisted of a 20% loss in body weight or showing at least two of the following clinical signs: lethargy, ruffled fur, hunched posture, labored breathing, dull or sunken eyes. Mice were euthanized when they reached endpoints via exposure to carbon dioxide (CO2) according to UHN facility guidelines.
1. Preparation of Glycerol Stocks for Long-term Storage
NOTE: This procedure describes how glycerol stocks of the EGD strain of L. monocytogenes are prepared from an original glycerol stock. Steps that have the potential to generate aerosols should be performed within a certified biosafety cabinet (BSC).
2. Determination of Growth Curve of L. monocytogenes in Day Culture
NOTE: This procedure describes how to generate the growth curve for L. monocytogenes that is used to estimate the colony forming units (CFU) for infection studies. All steps that have the potential to generate aerosols should be performed within a certified BSC.
3. Preparation of the Inoculum for Experimental Infection with L. monocytogenes
NOTE: This procedure describes the preparation of the infectious inoculum from a day culture that was started from an overnight culture (prepared in Procedure 2). All of these steps are performed in the BSC unless otherwise indicated.
4. Experimental Infection of Mice with L. monocytogenes
NOTE: This procedure describes how to infect mice with the inoculum prepared in Procedure 3 and how to verify the CFU delivered in the inoculum. Handling of mice and injections are performed in a BSC.
5. Preparing Heat-killed L. monocytogenes for Immune Studies
NOTE: All steps that have the potential to generate aerosols are performed within the BSC.
6. Measurement of IFN-γ Responses by NK and NKT Cells during Infection
NOTE: This procedure describes how to measure the IFN-γ responses by NK and NKT cells in mice at 24 hr after infection with 105 CFU of the L. monocytogenes. This dose is used because it induces robust IFN-γ responses by NK and NKT cells in the spleen24. Conduct all steps in the BSC. To help maintain cell viability, keep cells on ice whenever possible and use ice-cold buffers.
7. Measurement of Bacterial Load in the Spleen and Liver at the Time of Peak Infection
NOTE: All steps are performed within a BSC unless otherwise noted.
8. Effects of L. monocytogenes on IFN-γ Responses by CD4+ and CD8+ Cells
NOTE: This procedure describes how to measure IFN-γ production by splenic CD4+ and CD8+ T effector cells harvested at the time of the peak of the adaptive immune response (~ 7 d post-infection) using two methods: (1) flow cytometry to measure IFN-γ by CD4+ and CD8+ cells by intracellular cytokine staining, and (2) ELISA to measure total IFN-γ levels produced by splenocytes (includes all T cells). Procedures are performed within the BSC.
9. Measuring Mouse Survival to Endpoints after L. monocytogenes Infection
NOTE: This procedure describes the effect of an agent on mouse survival to endpoints post-infection with the modified LD50 dose of the pathogen. All these procedures are conducted in the BSC in the animal facility.
Figure 3 presents some typical flow cytometry staining of IFN-γ in splenic NK and NKT cells at 24 hr post-infection with 105 CFU of the pathogen. This figure also illustrates the gating strategy for the staining panel described in Table 2. Figure 4 shows some representative data that were obtained in one experiment where male mice were treated with the PPARα antagonist IS001 or vehicle control, infected with 105 CFU L. monocytogenes, and then analyzed for IFN-γ in NK and NKT cells after 24 hr. This figure shows that treatment with IS001 boosted IFN-γ responses by NKT cells, but not NK cells after infection with the pathogen. Figure 5 shows representative staining for IFN-γ in splenic CD4+ and CD8+ T cells at 7 days post-infection after re-stimulation ex vivo with heat-killed pathogen. This figure also shows the gating strategy for the staining panel described in Table 3. Figure 6 shows representative data that were obtained in one experiment where male mice were treated daily with the PPARα antagonist IS001 or vehicle control, infected with a sublethal dose of L. monocytogenes, and analyzed at 7 days post-infection. This experiment shows that treatment with IS001 enhanced IFN-γ responses by both CD4+ and CD8+ lymphocytes. Figure 7 shows representative data from a study that investigated the effect of the PPARα antagonist IS001 on mouse survival to endpoints after infection with the modified LD50 dose of the pathogen. Plotted is the percent survival of mice against time post-infection. This figure shows that treatment with IS001 increased the survival of male mice to endpoints. Together these data illustrate how this model can be applied to investigate the effects of new drugs or treatments on IFN-γ responses in vivo and to explore how these immune changes impact animal survival from infection.
Figure 1. Dissecting the Spleens from Infected Mice. This series of photos shows how to dissect the spleen from a dead mouse. (a) Lie the mouse on its right side and spray down the skin with 70% ethanol. (b) Using aseptic or sterile forceps and tough-cut scissors, incise the skin just below the bottom of the rib cage. (c) Spray down the exposed muscle layer with 70% ethanol. The spleen should be visible underneath the muscle layer (open arrow head). (d) Using aseptic or sterile forceps and fine scissors, incise the muscle layer to reveal the spleen. (e) Gently grab the spleen with the forceps and use fine scissors to cut the spleen away from surrounding the connective tissue. (f) Place the spleen in a 15 ml conical tube containing sterile 1x PBS. Please click here to view a larger version of this figure.
Figure 2. Counting Splenocytes using a Hemocytometer. (a) shows the central grid of the hemocytometer. (b) shows an enlarged view of the central grid that contains 25 large squares (that each contain 16 smaller squares). The five large squares used for counting are highlighted in grey (4 corner squares plus the center square in the central grid). (c) shows an enlarged view of one of the large grey squares. To determine the cell volume in 106/ml, first count all the viable cells within the five large grey squares. In the example shown, this count is 215. When counting, make sure to only count all of the clear (non-blue) cells, including those that are touching the double lines on the right and bottom of the grid. Do not count the cells touching the double lines on the left and top of the grid. Take the total five square count and divide it by 10 to obtain the number of cells in 106/ml. In the example, 215 divided by 10 is 21.5 x 106 cells/ml. Note that these calculations only work if you are counting 5 of the large squares as highlighted and dilute your cells 1:1 in trypan blue. Please click here to view a larger version of this figure.
Figure 3. Gating Strategy for Detection of IFN-γ Production in NK and NKT cells. First gate on lymphocytes on FSC-A by SSC-A plot. Then gate on those events that are on the diagonal on the FSC-H/FSC-A plot. These are the singlets. Then gate on live (AmCyan–) and CD8– cells. Then plot the tetramer staining against TCRβ. The NKT cells are within the double positive population and the NK cells are within the double negative population. Gate on the double positive cells, and plot NKp46 versus FSC. Gate on the NKp46 negative population, which are the NKT cells (this gate can be set by finding the point of division in the two populations from the NK cell plot). The NK cells are the tetramer– TCRβ–NKp46+ population. Within NK and NKT cell gates, the IFN-γ+ cells in the PE channel are identified after setting a gate based on the FMO control. Please click here to view a larger version of this figure.
Figure 4. Representative Data Obtained for the Frequencies of IFN-γ+ NK and NKT Cells at 24 hr Post-infection. In this experiment, male C57BL/6J mice (N = 3 – 4/group) were infected i.p. with 105 CFU of L. monocytogenes or were left un-infected. Mice were also administered the drug IS001 or vehicle (0.5% carboxymethyl cellulose) at the same time of inoculation and 12 hr later. Twenty-four hours after inoculation, mice were euthanized and the spleens were removed and were processed individually and stained for flow cytometry. Shown are the mean ± SEM frequency of IFN-γ+ cells in the NK (a) or NKT cell gates (b) in uninfected or infected mice after treatment with a vehicle or the drug IS001. *Indicates a difference (P < 0.05) from vehicle control by two-tailed T-test. Data are re-printed from31 with permission from the Journal of Immunology (volume 195, pp. 5189-5202, 2015). Copyright 2015. The American Association of Immunologists, Inc. Please click here to view a larger version of this figure.
Figure 5. Gating Strategy for Detection of IFN-γ Production in CD4 and CD8 cells. First gate on lymphocytes on FSC-A by SSC-A plots. Then gate on those events that are on the diagonal on the FSC-H/FSC-A plot. These are the singlets. Within this gate, gate on live (AmCyan–) CD45+ cells. Then gate on either CD8+ or CD4+ populations. Within each gate, the IFN-γ+ cells in the PE channel are identified by comparing the staining to the FMO control. Please click here to view a larger version of this figure.
Figure 6. Representative Data Obtained for the Frequencies of IFN-γ+ CD4+ and CD8+ T Cells at 7 Days Post-infection with L. monocytogenes (EGD strain). In this experiment, male C57BL/6J mice were infected i.p. with 2 x 104 CFU L. monocytogenes (N= 7/group) or were left uninfected (N= 3/group). Mice were also administered the drug IS001 or vehicle (0.5% carboxymethyl cellulose) twice daily starting on the day of inoculation. Seven days later, mice were euthanized and the spleens were removed and were processed individually for cell culture. Splenocyte mononuclear cells were stimulated for 24 hr with heat-killed L. monocytogenes with protein transport inhibitor added for the final 4 hr of culture. Cells were then stained for flow cytometry. Shown are the mean ± SEM frequency of IFN-γ+ cells in the CD4+ (a) or CD8+ cell gates (b) in uninfected or infected mice after treatment with a vehicle (0.5% carboxymethyl cellulose) or the drug IS001. * indicates a difference (P < 0.05) from the vehicle control counterpart as determined by two-tailed T test. Data are re-printed from31 with permission from the Journal of Immunology (volume 195, pp. 5189-5202, 2015).Copyright 2015. The American Association of Immunologists, Inc. Please click here to view a larger version of this figure.
Figure 7. Representative Data Obtained during an Experiment that Compared the Effect of a PPARα Antagonist 1S001 on Mouse Survival to Endpoints after Infection of Male C57BL/6J Mice with L. monocytogenes (EGD strain). In this experiment, male C57BL/6J mice (N = 10 mice/group) were infected i.p. with the modified LD50 dose of the pathogen (105 CFU) of L. monocytogenes. Mice were also administered the drug IS001 or vehicle (0.5% carboxymethyl cellulose) twice daily starting on the day of inoculation. Mice were followed daily for clinical signs and were euthanized if humane endpoints were met. Shown is the percent survival of mice to endpoints over time * indicates a difference in the survival between groups as determined by log-rank test (P < 0.05). Data are re-printed from31 with permission from the Journal of Immunology (volume 195, pp. 5189-5202, 2015). Copyright 2015. The American Association of Immunologists, Inc. Please click here to view a larger version of this figure.
Table 1: Shows Some Representative Calculations for Determining CFU in an Aliquot of Day Culture. In this example, an aliquot of day culture was taken and was diluted 1:1 with BHI media. The OD600 of this diluted sample was determined to be 0.84. In addition, a 100 µl aliquot was taken for CFU determination. This sample was diluted with 900 µl of BHI media (10-1) and was washed and resuspended in 1 ml BHI. A 10-fold dilution series of this sample was prepared (10-2 to 10-9) and diluted samples were plated on BHI agar plates (only values for 10-4 to 10-9 are shown). The next day colonies were counted. Only those plates that had colony numbers between 30-300 were considered for the calculation (i.e., 10-6 plate, highlighted in yellow). The number of colonies on this plate (70) was then divided by 0.1 (volume in ml plated) to get the CFU/ml of the diluted sample. This value was then multiplied by the dilution factor (106) to obtain the CFU/ml reading of the undiluted culture. TMTC = too many to count.
Table 2: Staining Panel for Detection of IFN-γ in NK and NKT Cells. Note that either compensation beads stained with the flow antibodies used in the panel or splenocytes stained with various fluorochrome versions of CD4 antibody clone GK1.1 can be used as single positive controls.
Table 3: Staining Panel for Detection of IFN-γ in CD4+ and CD8+ Cells. Note that either compensation beads stained with the flow antibodies used in the panel or splenocytes stained with various fluorochrome versions of CD4 antibody clone GK1.1 can be used as single positive controls.
Here we describe a protocol of how to carry out a basic experimental infection with the EGD strain of L. monocytogenes25 in male or female C57BL/6J mice. This protocol was set up for the purpose of studying the effect of a novel small molecule IS001 on IFN-γ production by innate and adaptive lymphocytes in vivo31. By monitoring bacterial clearance and survival post-infection, insights were gained into how these changes in IFN-γ impacted the host's ability to control the infection.
Critical Considerations in the Protocol
An important consideration in the design of this type of study is that each experiment be adequately powered and appropriately controlled. Due to biological variation in the immune response to infection (see Figures 4 and 6), it is recommended that N = 4 – 5 mice per group should be used for the initial immune studies. If after these studies there is a trend in the data, but no significant difference apparent between groups, a power calculation could be done to determine the least number of animals required in subsequent studies to achieve statistical significance. Regarding controls, it is important to include uninfected controls for determination of baseline IFN-γ responses for immune studies and vehicle controls to help distinguish the effect of the treatment from the stress associated with administering the treatment. Another important consideration is the timing of treatment. Since the innate response to L. monocytogenes is very rapid, it is recommended that the first treatment be administered on the day prior to, or at the same time as, inoculation in order to ensure that therapeutic levels of the reagent be achieved prior to the initiation of the innate immune response.
Yet another important consideration is the dose of the pathogen to be used for infection. A sublethal dose is recommended for measurement of bacterial load, since it increases the chance that the pathogen will be concentrated within the spleen and liver, allowing for the more accurate enumeration of the bacteria. A sublethal dose is also recommended for enumerating IFN-γ responses by adaptive lymphocytes to ensure that animals do not succumb to listeriosis prior to the time of peak T cell expansion. In contrast, it is recommended that a higher infectious dose be used for measurement of the early NK and NKT cell response at 24 hr in order to maximize the IFN-γ production by these cells.
The classical LD50 is the dose of pathogen that results in 50% lethality of mice. Since death was not an acceptable endpoint at our institution and since many symptoms of listeriosis can predict whether an animal is likely to succumb to an infection, we used a defined list of clinical signs instead of death as an endpoint in our studies. Using this method, it was determined that the modified LD50 was 105 CFU for 8-week-old male and 1.5 x 105 CFU for 8-week-old female C57BL/6J mice31. These LD50 doses were determined by measuring the percent survival of mice to endpoints in step-wise dose-escalation studies (N = 5 studies in total) that each contained N = 8 mice per group (e.g., mice were infected first with 10,000 CFU, then a second batch with 20,000 CFU, etc.). The LD50 calculation was determined from a regression plot of the log (CFU) (x-axis) versus the probit of the percent survival values (y-axis) (website: userwww.sfsu.edu/efc/classes/biol710/probit/ProbitAnalysis.pdf).
Note that the modified LD50 dose determined in our lab may differ from that in another lab even when infecting mice with the same strain of L. monocytogenes. Part of this variability may relate to the subjective nature of monitoring clinical signs of listeriosis compared to the more absolute endpoint of death. Additional variability can result from differences in environmental factors such as mouse diet or the microbiota or differences in the preparation of inoculum between labs. Thus, it is recommended that prior to embarking on any survival studies, a pilot study be performed where female mice (N = 8 mice/group) are infected with 1.5 x 105 CFU of the same strain of L. monocytogenes as used in this study and symptoms monitored to determine if this dose indeed results in 50% survival to endpoints. If survival is lower or higher than 50% at this CFU, step-wise dose escalation or dose de-escalation studies could be performed to quickly narrow in on the LD50 dose.
Another important consideration is the strain or substrain of mice used for infection studies. This protocol describes infection of the commonly-used inbred mouse strain C57BL/6J. This strain is well-suited for measurement of IFN-γ responses since this mouse is considered to be a Th1-prone strain33 and as a result, is relatively resistant to L. monocytogenes infection (compared to Th2-prone mouse strains such as BALB/c)34,35. Adapting this protocol to other mouse strains will require knowledge of the infectious dose of the pathogen for the particular strain. It is also recommended to use mice of the same age, sex and vendor as outlined in this protocol in order to reduce the amount of trouble-shooting involved in setting up the model. For example, C57BL/6 mice ordered from one vendor (e.g., C57BL/6J) can exhibit genetic differences than C67BL/6 mice ordered from another vendor (e.g., C57BL/6NTac)36. In addition, the intestinal microbiota differs between C57BL/6 substrains obtained from different vendors, which can influence the balance of Th1 and Th17 responses in the mouse37.
Potential Modifications to Technique
Mice are most commonly inoculated i.p. or intravenously as opposed to the natural route of infection in humans, which is through the gastrointestinal tract. Oral infections are less common because standard strains of L. monocytogenes inefficiently infect the intestinal epithelium of mice38. This is because there is a single amino acid change in the sequence of mouse E-cadherin from human E-cadherin that results in loss of recognition of E-cadherin by the listerial invasion protein, internalin A (InIA)39. To overcome this barrier, researchers use mice that are transgenic for human E-cadherin protein or use listeria that have been engineered to express a mutated sequence of InIA (InIAmut) that binds to mouse E-cadherin with the same affinity as WT EGD for human E-cadherin40. Thus, one potential modification of this technique is to infect mice via the oral route. The reader is referred to another JoVE publication that describes oral inoculation methods38. Note that altering the mode of infection will affect the infectious dose as well as the kinetics of dissemination of the pathogen.
This protocol describes using heat-killed listeria to elicit IFN-γ production by CD4+ and CD8+ T cells. Heat-killed L. monocytogenes was chosen as a stimulus in our studies, because this antigen is inexpensive and because our lab was primarily interested in CD4+ T cell responses to the pathogen. One limitation is that heat-killed bacteria do not efficiently prime CD8+ T cell responses either in vitro41 or in vivo42,43 infection. Thus, the CD8+ T cell IFN-γ production that we observed by splenocytes harvested at the peak of infection (i.e., Figure 6) likely is in response to the residual live bacteria present in the splenocyte cultures or was elicited as a result of cytokine-induced cytokine release41. As an alternative to heat-killed listeria, one could also elicit IFN-γ responses ex vivo by exposing T cells to peptides encoding epitopes on listerial proteins. Indeed, immunodominant MHC Class II-restricted epitopes for listeriolysin O and the p60 hydrolase and have been described for C57BL/6 and BALB/C mice and immunodominant MHC Class I epitopes have been described for BALB/c44. Yet another approach is to infect mice with strains of L. monocytogenes that have been engineered to express model antigens such as ovalbumin or viral antigens in order to take advantage of existing MHC Class I- and MHC Class II-tetramer reagents to enumerate antigen-specific T cells in infected mice45,46.
Other Limitations of the Protocol
Another limitation of this model is that it only measures IFN-γ production by immune cells in the spleen. In addition to the use of tetramers to enumerate antigen specific T cells (of ovalbumin-expressing variants of L. monocytogenes), flow cytometry staining panels described here could be easily modified to measure the production of other cytokines such as TNF or IL-2 or effector molecules that participate in CD8 T cell or NK-mediated killing of the pathogen such as perforin or granzyme B. In addition, this protocol could also be adapted to examine IFN-γ produced by immune cells in the liver.
Future Applications
Once this protocol is mastered, it can serve as a simple in vivo model to screen the effects of various agents or genes on Th1 and cellular immunity.
The authors have nothing to disclose.
Development of this protocol was supported by an operating grant from CIHR (MOP97807) to SED.
Brain Heart Infusion Broth, Modified | BD | 299070 | any brand should be appropriate |
Agar | BD | 214010 | any brand should be appropriate |
Triton X-100 | Sigma-Aldrich | X100 | any brand should be appropriate |
1xPBS | Sigma | D8537 | any brand should be appropriate |
TissueLyser II | Qiagen | 85300 | any brand should be appropriate |
Ammonium Chloride (NH3Cl) | any brand should be appropriate | ||
KHCO3 | any brand should be appropriate | ||
Na2EDTA | any brand should be appropriate | ||
RPMI 1640 | Gibco | 22400089 | any brand should be appropriate |
Fetal Bovine Serum | Gibco | 12483 | Before use, heat-inactivate at 56 °C for 30 min |
L-glutamine | Gibco | 25030 | any brand should be appropriate |
Non-essential amino acids | Gibco | 11140 | any brand should be appropriate |
Penicillin/Streptomycin | Gibco | 15140 | any brand should be appropriate |
GolgiStop Protein Transport Inhibitor (containing Monensin) | BD | 554724 | Use 4 μl in 6 ml cell culture |
16% Paraformaledehye | Electron Microscopy Sciences | 15710 | Dilute to 4% PFA in ddH20 or 1xPBS |
10 x Perm/Wash buffer | BD | 554723 | Dilute 10x in ddH20 |
Fc block, Anti-Mouse CD16/CD32 Purified | eBioscience | 14-0161 | Dilute 1:50 |
Fixable Viability Dye eFluor 506 | eBioscience | 65-0866 | Dilute 1:1000 (we have also used viability dyes from Molecular Probes) |
anti-Mouse CD4-PE-Cy5 (GK1.5) | eBioscience | 15-0041 | Manufacturer recommends a certain test size; however this should be titrated before use. |
anti-Mouse CD8-FITC (53-6.7) | eBioscience | 11-0081 | Manufacturer recommends a certain test size; however this should be titrated before use. |
PBS57/mCD1d tetramer-APC | NIH Tetramer Core Facility | N/A | Obtained as a gift from the facility |
anti-Mouse TCRβ-PE-Cy7 (H56-597) | eBioscience | 25-5961 | Manufacturer recommends a certain test size; however this should be titrated before use. |
anti-Mouse NKp46-APC-eFluor780 (29A1.4) | eBioscience | 47-3351 | Manufacturer recommends a certain test size; however this should be titrated before use. |
anti-Mouse CD45 PE-Cyanine7 (30-F11) | eBioscience | 25-0451 | Manufacturer recommends a certain test size; however this should be titrated before use. |
anti-Mouse IFN gamma-PE (XMG1.2) | eBioscience | 12-7311 | Manufacturer recommends a certain test size; however this should be titrated before use. |
OneComp eBeads | eBioscience | 01-1111 | Manufacturer recommends a certain test size; however this should be titrated before use. |
Mouse IFN gamma ELISA kit | eBioscience | 88-7314 | Used for measuring the interferon gamma in the culture supernatant |
50 mL vented tubes for culture | Used for culturing the bacteria, any brand should be appropriate | ||
1.5 ml microcentrifuge tubes | any brand should be appropriate | ||
bacterial petri dishes | any brand should be appropriate | ||
2 ml cyrovials | any brand should be appropriate | ||
UV spectrometer | any brand should be appropriate | ||
safety engineered needles | any brand should be appropriate | ||
C57BL6/J | Jackson laboratories | Stock#000664 | Order for arrival at 7 wks |
Bleach | For decontamination | ||
70% Ethanol | For decontamination | ||
Glass beads | any brand should be appropriate | ||
Centrifuge | rotor, buckets, bucket covers. | ||
Microcentrifuge | any brand should be appropriate | ||
Sterile Glycerol | any brand should be appropriate | ||
Pipette Tips | any brand should be appropriate | ||
Pipette | any brand should be appropriate | ||
Surgical instruments | any brand should be appropriate | ||
70 micron strainers | any brand should be appropriate | ||
3 ml syringe | any brand should be appropriate | ||
Pipette gun | any brand should be appropriate | ||
Filtration Units | any brand should be appropriate | ||
Trypan Blue | Dilute 1 to 9 in ddH20, any brand should be appropriate | ||
Hemocytometer | any brand should be appropriate | ||
Round bottomed plates | any brand should be appropriate | ||
FACs tubes | BD | ||
BD LSR II | BD | Any flow cytometer could be used for acquisition that has an appropriate laser configuration and filter set to discriminate the fluorochormes | |
Flowjo software | Treestar | Used for data analysis. Other types of data analysis software will also be appropriate | |
Multichannel pipettor (0-300 µl) | Eppendorf | Used for washing cells and adding antibodies during flow cytometry staining | |
Acetic Acid | Used for washing glass beads, any brand should be appropriate | ||
Microbank Bacterial Preservation System | Pro-lab Diagnositics | Used as an alternative to glycerol stocks for long-term storage of bacteria |