Long-term cultured interferon-γ enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.
Effector and memory T cells are generated through developmental programing of naïve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-γ. Interferon-γ release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-γ ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.
Effector and memory T cells are generated through developmental programing of naïve CD4+ T cells after antigen recognition. Differentiation of naïve CD4+ T cells into cytokine producing cells requires pathogen recognition by innate immune cells, antigen presentation to T cells, co-stimulation and transcriptional changes resulting in polarized cytokine production. For example: antigen presenting cells produce interleukin (IL)-12 in response to intracellular pathogens, which, together with antigen recognition, promotes differentiation of T cells into T helper 1 (Th1) cells1,2 by signaling through signal transducer and activator of transcription 4 (STAT4) and T-box expressed in T cells (T-bet), leading to cell activation, IL-2 production, clonal expansion and interferon (IFN)-γ production3,4. If the infection is controlled, up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime5. Sallusto et al.6, revealed two functionally distinct subsets of memory T cells in humans based on the expression of lymph node homing receptors. Central memory T cells (Tcm) express C-C chemokine receptor (CCR)-7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Tcm have limited effector function, a low activation threshold and retain high IL-2 production and proliferative capacity. Effector memory T cells (Tem) express CD45RO, lack CCR7 and display receptors for homing to peripheral or inflamed tissues. Effector cells do not express either CCR7 or CD45RO but promptly produce effector cytokines, such as IFN-γ, upon antigen recognition.
IFN-γ release assays (IGRA) are used for the diagnosis of bovine and human tuberculosis7. Mycobacterium bovis is the principal agent of bovine tuberculosis (bTB) while human cases of tuberculosis are caused mainly by Mycobacterium tuberculosis. With these tests, whole blood or peripheral blood mononuclear cells (PBMC) are stimulated with mycobacterial antigens for 16 to 24 hr and IFN-γ production within the supernatant is measured by ELISA or through detection of cells producing IFN-γ using ELISPOT techniques. As a result of the brief stimulation period (i.e., 16 to 24 h) and rapid cytokine production, ex vivo assays detect primarily effector and Tem responses. This has been confirmed by flow cytometric analysis of cell populations in these cultures 8-10.
Ex vivo IFN-γ responses are routinely included within the immune response panel evaluation of tuberculosis vaccines, including those used to evaluate responses by cattle. Most effective bovine tuberculosis vaccines elicit specific IFN-γ responses, but not all vaccines that induce IFN-γ responses are protective. Also, levels of IFN-γ elicited by vaccination, as measured before infection, do not necessarily correlate with protection. For instance, different BCG strains may have different capacities to induce ex vivo IFN-γ response, in spite of similar protection levels11. Thus, ex vivo IGRAs are valuable for tuberculosis diagnosis and for accessing vaccine immunogenicity; however, their use as predictors of vaccine efficacy is limited. Tcm responses to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus (SIV) infection in non-human primates12,13, tuberculosis in mice14 and malaria in humans15,16.
After an effective immune response resulting in pathogen clearance, Tcm are maintained and provide protection to an eventual second infection by the same agent. A notable exception to this scenario is M. tuberculosis infection of humans in which patients receiving curative anti-mycobacterial therapy are susceptible to re-infection17,18. Additionally, events governing immunological memory during chronic infections, wherein the antigenic stimulation persists, are not well understood19. During chronic infections, such as with human immunodeficiency virus (HIV) and tuberculosis, a significant Tcm response is associated with a favorable outcome (e.g., latency with tuberculosis and subclinical disease with HIV)20,21. Several studies with mice and humans have demonstrated that long-term cultured IFN-γ ELISPOT assays measure Tcm responses16,20-22. With this assay, PBMCs are cultured in decreasing concentration of antigen for 10 to 14 days, allowing effector responses to peak and wane; facilitating Tcm to differentiate and expand within the culture.
Long-term cultured IFN-γ ELISPOT assays have also been used in veterinary research, yet the phenotype of responding cells has been difficult to assess due to a lack of critical reagents, especially an antibody to CCR7. Effective bovine tuberculosis vaccines [e.g. using a single dose of M. bovis Bacille Calmette Guerin (BCG), BCG followed by viral-vectored Antigen 85A subunit vaccine, or attenuated M. bovisΔRD1] elicit long-term cultured IFN-γ ELISPOT responses following vaccination that correlate with protection (i.e., lower mycobacterial burden and decreased TB-associated pathology) against subsequent challenge with virulent M. bovis23,24. Furthermore, the numbers of antigen-specific IFN-γ-secreting cells within long-term PBMC cultures are higher at 12 months but decrease at 24 months after BCG vaccination of neonatal calves, correlating with the degree of protection detectable post M. bovis challenge25. In this scenario, the cultured IFN-γ ELISPOT is an important tool for predicting vaccine efficacy, providing a means to prioritize vaccine candidates for high cost efficacy trials. Additionally, cultured ELISPOT techniques can be adapted for various hosts, pathogens and cytokines by altering antigens or antibodies for a variety of purposes in different research fields.
1. Prepare the Following Solutions
2. Long-term Cultured Cells (14 Day Protocol)
(Day one)
(Days three and seven)
(Days 10 and 12)
3. ELISPOT Assay (Three Day Protocol)
(Day one (12 th day of Long-term culture protocol))
(Day two (13 th day of the long-term culture protocol))
4. Short-term Cell Culture and Adherent Cell (APCs) Isolation
5. Cultured Cells
6. Plating Fresh and Cultured PBMCs
Day three (14 th day of the Long-term culture protocol)
7. Plates Reading and Data Analysis
Approximately one-month post-aerosol infection with M. bovis (104 colony-forming units), PBMCs from infected (n=8) and control animals (n=8) were cultured in the presence of antigens and IL-2 for 13 days. Development of Tcm responses after infection was determined using IFN-γ ELISPOT assay. Representative Tcm (in the presence or absence of APCs) and exvivo IFN-γ ELISPOT responses from infected animals (Three animals) and a non-infected animal (one animal) are shown in Figure 1. A successful long-term IFN-γ ELISPOT assay results in cells producing IFN-γ (Spot-Forming Cells, SFC) under stimulated conditions and near absence of a response from non-infected animals and under non-stimulated conditions. Also, a strong T cell response should occur in response to PWM. Specific responses to M. bovis were assessed by PPD-B or rESAT-6:CFP10 antigenic stimulation. As shown in Figure 1, robust Tcm and ex vivo responses to PPD-B and rESAT-6:CFP10 were detected from all three M. bovis-infected animals. Minimal to no Tcm and ex vivo responses were detected from the control animal. Also, the presence of APCs was required for optimal Tcm responses by infected animals, as demonstrated by greatly reduced responses by long-term cells cultured in the absence of autologous APCs. IFN-γ response by PBMCs from M. bovis infected and non-infected animals are presented in Figure 2. The number of SFCs from each animal was calculated as the average number of SFCs in duplicate samples in response to PPD-B minus the respective response to media alone. Responses were estimated for 106 cells. Responses differed (P < 0.05) based upon infection status, presence or absence of APCs and culture duration (i.e., long-term culture versus ex vivo culture).
Acquiring image of plates | |
1. | Turn on machine |
2. | Open “Immuno Capture” Version 6.3 software. |
3. | Step 1: select plate type. |
4. | Step 2: load plate. |
5. | Step 3: select scanning options. |
6. | Step 4: start scanning. |
7. | Obtain overview image of plate. |
8. | Eject plate. |
9. | Quit “Immuno Capture” software. |
Counting the spot forming units | |
1. | Open “Immuno Spot Capture” Version 5.0 software. |
2. | Select object type: normal. |
3. | Select counting module: smart count. |
4. | Step 1: load plate. |
5. | Step 2: define counting parameters: test accuracy of spot recognition on wells with different spots. |
6. | Start auto count. |
Quality control | |
1. | Open “Immunospot Capture” Version 5.0 software. |
2. | Select counting module: quality control. |
3. | Step 1: load plate. |
4. | Step 2: Analyze highlighted wells individually. |
5. | Finish quality control. |
Table 1 – AutoImmun Diagnostika ELISPOT reader image acquisition and cell counting procedure.
Figure 1. Image of wells from a long-term cultured IFN-γ ELISPOT assay. Cultured IFN-γ ELISPOT assay was performed approximatelyone month after challenge with virulent M. bovis (three animals). Non-infected animals were included as controls (one animal). Long-term cell lines were generated by stimulating PBMC with a cocktail of recombinant Ag85A (1 µg / ml), TB10.4 (1 µg / ml), rESAT-6:CFP10 (1 µg / ml) and PPD-B (5 µg / ml) for 13 days followed by transfer to ELISPOT plates in the presence or absence of autologous APC. Short-term cells consisted of PBMC isolated on day 13 and plated directly into ELISPOT plates. Long-term and short-term cells were stimulated with PPD-B (10 µg / ml), rESAT-6:CFP10 (1 µg / ml), medium alone or pokeweed mitogen (5 μg / ml) for 24 h.
Figure 2. Representative results from a long-term cultured IFN-γ ELISPOT response to M. bovis purified protein derivative (PPD-B). Cultured IFN-γELISPOT assay was performed approximatelyone month after challenge with virulentM. bovis (eight animals). Non-infected animals were included as controls (eight animals Long-term cells were generated by stimulating PBMC with a cocktail of recombinant Ag85A (1 µg / ml), TB10.4 (1 µg / ml), rESAT-6:CFP10 (1 µg / ml) and PPD-B (5 µg / ml) for 13 days followed by transfer to ELISPOT plates in the presence or absence of autologous APCs. Short-term cells consisted of PBMC isolated on day 13 and plated directly into the ELISPOT plate. Long-term and short-term cells were stimulated with PPD-B (10 μg / ml) or medium alone for 24 h. Specific responses from each animal (SFC / 106 cells) are presented as response to PPD-B (average of duplicate samples) minus the response to media alone.
IFN-γ production in long-term cultured ELISPOT assays is generally due to Tcm in humans, but how this response develops in culture is poorly understood. Whether Tcm are present in circulation and expand in vitro, or if the Tcm responses result from differentiation of effector and Tem cells into Tcm during culture is not known. However, studies with samples from humans have found that CD4+ T cells from ex vivo and long-term cultured IFN-γ ELISPOT assays have unrelated epitope specificities, implying that Tcm responses do not develop from effector cells28. Obviously, the duration of the response may vary, but if pathogen clearance is achieved, eventually both ex vivo and Tcm responses decline. Also, responses of long-term cultures measured early and long after antigenic priming (when the effector response is lower) is shown to correlate, indicating that circulating Tcm populations early and long after antigenic priming remains related in vivo. On the other hand, the magnitude of the ex vivo response does not appear to be related to the magnitude of the memory response29. These results suggest that long-term cultured IFN-γ ELISPOT responses result from in vitro expansion of Tcm and an associated waning of effector responses in the sample, rather than the differentiation of effector cells into Tcm phenotype.
With cattle, the relative contribution of effector and memory subsets to responses detected by the long-term IFN-γ ELISPOT assays is not known. Recent studies indicate a correlation between vaccine elicited long-term cultured IFN-γ ELISPOT responses and protection against subsequent experimental infection with M. bovis23-24. It has been reported that weak long-term IFN-γ ELISPOT responses to vaccination are associated with the absence of protection30. Both live and killed vaccines induce similar ex vivo IFN-γ ELISPOT responses, but vaccination with live BCG elicits stronger long-term cultured IFN-γ ELISPOT responses than do killed vaccine preparations. Interestingly, live vaccines are protective while killed formulations fail to provide protection against challenge with virulent tuberculous mycobacteria 31-32, 33.
Assessment of Tcm responses is also feasible by sorting these cells from PBMC. Direct enrichment of Tcm from PBMC, however, requires expensive devices, highly trained personal and is difficult as these cells are not numerous in the blood stream. Long-term culture of PBMC provides enrichment of Tcm over Tem and effector cells without expensive devices; however, because these T cell populations are expanded in vitro they may be less representative of in vivo memory responses. It is possible to access IFN-γ memory responses (using the long-term culture or Tcm sorting strategies) by assays other than the ELISPOT such as: cytokine ELISAs34, cytokine bead arrays (CBA), intracellular staining (ICS), or cytokine protein arrays (CPA). These methods; however, are generally less sensitive than ELISPOT assays35.
An advantage of ELISPOT is its ability to detect the immediate capture of the cytokine shortly after its release preventing dilution in the supernatant and degradation by enzymatic cleavage or cytokine uptake by other cells. ELISPOT assays detect single cells producing cytokines providing precise results even in low signal to noise scenarios (i.e., low specific responses)35. Also, with ICS, detection of cytokines prior to release may result in false identification of cells producing cytokines (e.g., due to post-translational modulation before or during the secretory process)34. The transport inhibitors employed with ICS assays to minimize cytokine secretion during antigen stimulation (known as Golgi stop proteins) limit the duration of cell stimulation due to the cell toxicity of these proteins ultimately impacting cytokine production35.
With that said – CBA, CPA and ICS are useful techniques for measuring multiple cytokines simultaneously and / or for determining cell surface marker expression (i.e., with ICS). Therefore, these methods may be used in combination with the IFN-γ ELISPOT assay36. While previous bovine TB vaccine efficacy studies measured Tcm responses via ELISPOT assay, the detection of Tcm responses by other techniques will likely yield comparable results. Importantly, the ELISPOT assay is less expensive and simpler to perform than CBA, CPA and ICS analysis techniques34. Manual counting of the SFC is an alternative to automated counting, and ELISPOT plates can be stored at RT for long period of time with minimal quality loss, before or after spot counting37.
The long-term cultured IFN-γ ELISPOT response has been applied to assess memory responses by human, and cattle when evaluating tuberculosis vaccine responses14-16,31-32,33. Tcm responses are also assumed to play significant roles in host responses to several other infectious agents of cattle, such as: Mycoplasma mycoides subsp. mycoides42, Anaplasma marginale38 and bovine respiratory syncytial virus39. Potentially, the long-term ELISPOT assay might be adapted for other animal species, cytokines and infections. These broader applications will be especially useful for veterinary immunology applications due to current limited availability of specific reagents.
Tcm responses are crucial for protection against several infections, but measuring them early after infection / immunization (for disease outcome prediction or vaccine efficacy) may be cumbersome. Analysis of the immune response under ex vivo and short antigenic stimulation conditions will more often represent effector responses, due to the overlap of memory and effector responses, especially during the beginning of immunological memory formation. In the context of chronic diseases in which the antigen load is continual, ex vivo responses will assess effector cell responses or a combination of memory and effector cell responses. The long-term cultured IFN-γ ELISPOT assay, described here, likely enables the measurement of Tcm responses rather than effector T cell or combined CD4+ T cell responses. In summary, the long-term cultured IFN-γ ELISPOT assay provides a valuable approach for estimating T cell memory responses and has been employed successfully to evaluate memory responses of several species to a variety of infections agents12-16, 20-25,29,31,33,34,38,39.
The authors have nothing to disclose.
Research was supported by USDA, ARS Cris: 3625-32000-104 and Agriculture and Food Research Initiative Competitive Grant no. 2011-67015-30736 from the USDA National Institute of Food and Agriculture. We thank Jessica Pollock, Emma Frimml-Morgan, Shelly Zimmerman, Kristin Bass, Bruce Pesch, Molly Stafne, Allen Jensen, and Tracy Porter for their excellent technical assistance as well as Rebecca Madison, Doug Ewing, Katie Pille, Jay Steffen, David Lubbers, Robin Zeisness, and David Panthen for the excellent care and handling of animals.
Sodium citrate (dihydrate) | Various | ||
Citric acid (monohydrate) | Various | ||
Dextrose | Various | ||
ELISPOT PVDF plate | Various | ||
Vectastain ABC – AP KIT Standard | Vector Laboratories | AK-5000 | |
Vector Blue Alkaline Phosphatase Substrate Kit | Vector Laboratories | SK-5300 | |
Ag85A | Lionex | LRP-0004.3 | 23, 24, 25, 37 |
TB 10.4 | Lionex | LRP-0061.6 | 23, 24, 25, 37 |
PPDb | Prionics AG | 7600055 | |
rESAT-6:CFP10 | Kind gift Dr. Minion, Iastate | 23, 24, 25, 27, 37 | |
Mouse anti-bovine IFN-γ Clone CC302 | Serotec | MCA1783B | 23, 24, 25, 27, 37 |
Mouse anti-bovine IFN-γ Biotinylated Clone CC330 | Serotec | MCA2112 | 23, 24, 25, 27, 37 |
Mouse anti-bovine CD4 Clone CC8 | Serotec | MCA1653GA | |
Mouse anti-bovine CD45RO Clone IL116A | Serotec | MCA2434GA | |
Rat anti-human CCR7 Clone 3D12 | Abcam | ab95665 | |
Mouse anti-bovine IFN-γ-Pe Clone CC302 | Serotec | MCA1783PE | |
Goat anti- mouse IgG2a- Alexa Fluor 350 | Invitrogen | A-21130 | |
Goat anti-mouse IgG3 APC-CY7 | SouthernBiotechnology | 1080-193 | |
Goat anti-rat IgG-APC | Invitrogen | A10540 | |
BD Cytofix/Cytoperm™ | BD Biosciences | 554714 | |
BrefeldinA | Sigma-Aldrich | B7651 | |
Pokeweed Mitogen | Sigma-Aldrich | L8777 | |
Recombinat human Interleukin 2 | Sigma-Aldrich | I7908 |