- Email: [email protected]
In situ tetramer staining
Journal of Immunological Methods 268 (2002) 29 – 34 www.elsevier.com/locate/jim
Review
In situ tetramer staining Pamela J. Skinner, Ashley T. Haase * Department of Microbiology and Great Lakes Center for AIDS Research, University of Minnesota Medical School, MMC 196, 420 Delaware St. SE, Minneapolis, MN 55455, USA Received 1 October 2001; accepted 14 January 2002
Abstract The development of MHC tetramer staining has opened the doors to multiple avenues of new research [Science 274 (1996) 94]. In this review, we will discuss the development and application of in situ MHC tetramer (IST) staining. We describe two independently developed IST staining methodologies and discuss current uses, limitations, future uses and the interesting biology revealed by the use of IST staining. D 2002 Elsevier Science B.V. All rights reserved. Keywords: In situ tetramer staining; Vibratome; HIV; SIV
1. Development of the technique 1.1. Indirect IST staining The development of MHC tetramer staining has revolutionized our ability to study antigen specific T cells (Altman et al., 1996), but until recently, its use has been limited to ex vivo studies. We sought to expand this methodology and describe here the successful development of a technique that extends tetramer staining to visualize antigen specific CD8 + T cells in tissues with their spatial relationship to other cells intact. In order to expedite the development of an in
Abbreviations: IST staining, in situ tetramer staining; TCR, T cell receptor; HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; ISH, in situ hybridization; APC, allophycocyanin; GFP, green fluorescent protein. * Corresponding author. Tel.: +1-612-624-4442; fax: +1-612626-0623. E-mail address: [email protected] (A.T. Haase).
situ tetramer (IST) staining technique, we utilized T cell receptor (TCR) transgenic mice (Sha et al., 1988; Hogquist et al., 1994). In these mice, the vast majority of T cells are identical. Our strategy involved making MHC class I tetramers with ExtraAvidin-FITC (Sigma) and then using FITC as an epitope to amplify the tetramer signal. Because we thought that it would be important to maintain the mobility of TCRs to interact with tetramers, we initiated studies using fresh unfixed tissue sections. Using a vibratome (TPI), we generated 200 Am thick fresh spleen sections. The sections were stained in a solution containing 0.5 Ag/ml tetramers, CD8 antibodies, 2% normal goat serum and PBS and incubated at 4jC overnight. The sections were then fixed, and the signal from the tetramers was amplified by incubating the sections with rabbit anti-FITC antibodies followed by incubation with goat-anti-rabbit conjugated to Cy3 (or other fluorophores). This strategy was successful and an example of these results is shown in Fig. 1 (Skinner et al., 2000). As negative controls, we either stained sections with MHC tet-
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 1 9 7 - 7
30
P.J. Skinner, A.T. Haase / Journal of Immunological Methods 268 (2002) 29–34
Fig. 1. MHC tetramers used to stain fresh spleen sections from 2C transgenic mice. (A – C) A section that was stained with Kb/SIY tetramers (A, red) and anti-CD8 clone 53-6.7 antibodies (B, green). (C) Images from (A) and (B) merged. (D – F) A section that was stained with negative control Kb/OVA tetramers (D, red) and anti-CD8 clone 53-6.7 (E, green). (F) Images from (D) and (E) merged. The confocal Z scans shown in (A) and (D) were collected using the same parameters with a 60 objective. The spleen used in this experiment was stored in PBS at 4j for 24 h prior to sectioning and staining. Reprinted with permission from Skinner et al. (2000). Copyright 2000. The American Association of Immunologists.
ramers loaded with an irrelevant peptide or we stained spleen sections from a different line of TCR transgenic mice. We then tested the method in a more realistic system where only a small fraction of the CD8 + T cell population is antigen specific. We analyzed spleen sections from a wild type mouse in which transgenic T cells had been adoptively transferred (Kearney et al., 1994; Schmidt and Mescher, 1999) (Fig. 2). Tetramer reactive CD8 + T cells were counted and the percentage of CD8 + cells that were tetramer positive was determined. Cells from half of the same spleen were analyzed by flow cytometry and the percentage of CD8 + T cells stained with tetramers was determined. Both the tetramer stained sections and the flow cytometry data showed that f 1% of the CD8 + T cells stained with tetramers. Thus, IST staining is equivalent in specificity and in sensitivity to flow cytometry.
In order to analyze archived tissue samples, we next determined whether IST staining would work with fixed or frozen tissues. Using spleens from TCR transgenic mice (Sha et al., 1988; Hogquist et al., 1994), we could detect tetramer staining in spleen tissue that had been lightly fixed in 2% formaldehyde for 20 min, or fixed in 50% methanol and 50% acetone for 5 min at 20 jC. However, the intensity of staining was much weaker and the background higher than unfixed fresh tissue sections. Antigen specific CD8 + T cells were also detected in frozen tissue sections. Frozen sections, 10 Am thick, on slides, were kept wet throughout the staining procedure, and during washing steps many sections were lost. The remaining stained sections showed tetramer positive cells, but the staining was not nearly as intense compared to fresh tissue. Moreover, in principle 20 times as much tissue can be examined for antigen specific CD8 + T cells in thick fresh sections.
P.J. Skinner, A.T. Haase / Journal of Immunological Methods 268 (2002) 29–34
31
been described in (Haanen et al., 2000). They also used fresh spleen sections from TCR transgenic mice and sections from spleens with adoptively transferred antigen specific T cells (Mamalaki et al., 1993; Toes et al., 1996) and exploited the high intensity signal emitted in MHC class I tetramers labeled with allophycocyanin (APC) to directly stain antigen specific T cells. Tetramer stained cells in the tissue sections were quantitated and compared to quantities obtained by flow cytometry and found to be similar. Comparison of the two described IST staining methods showed advantages and disadvantages for each. The intensity of staining using the indirect method, with amplification, is greater than the direct staining method, measured by the confocal laser power needed for detection (maximum confocal laser power used to see APC labeled tetramers, P. Skinner, unpublished). Because of the weaker signal, we were not able to sample as deeply into the tissue. The major advantage we have found with the direct staining method is lower background staining and the ability to more easily colabel with antibodies to other interesting proteins in the tissue.
2. Limitations
Fig. 2. Adoptively transferred T cells detected in situ using MHC tetramers. Fresh spleen sections from a wild type mouse that 2C transgenic mouse lymphocytes were adoptively transferred were stained with (A) Kb/SIY tetramers (red) and (B) anti-CD8 antibodies (green). (C) Images from (A) and (B) merged. Reprinted with permission from Skinner et al. (2000). Copyright 2000. The American Association of Immunologists.
For these reasons, we have subsequently focused on and recommend using fresh tissues for IST staining. 1.2. Direct IST staining Another IST staining method using MHC class I tetramers to stain antigen specific CD8 + T cells has
IST staining is limited in the same ways that exvivo MHC tetramer techniques are limited. The technique requires cloned and characterized MHC molecules and MHC matched tissue. These are sometimes difficult to obtain. Another limitation is that individual MHC tetramers only detect a subset of the total pool of antigen specific T cells directed against specific pathogens. Given that an individual can have up to six different MHC class I alleles, and pathogens are comprised of innumerable immunogenic peptides, using MHC tetramers to study the entire repertoire of CTL response to a pathogen is daunting. However, increasing numbers of MHC alleles from multiple species are rapidly being cloned and characterized making broad studies of CTLs more feasible. An additional limitation with IST staining is the requirement of fresh tissue specimens for optimal results. The requirement for fresh tissues will likely also require, in the short term, new prospective studies. Fresh tissues must be processed immediately. However, they can be obtained at a distant site and shipped on ice and
32
P.J. Skinner, A.T. Haase / Journal of Immunological Methods 268 (2002) 29–34
sectioned and stained the following day without significant loss of signal.
3. Summary of current practices and examples of interesting biology revealed Our primary objective in developing an IST staining technique was to use IST staining to learn more about human immunodeficiency virus (HIV) specific CD8 + T cells in humans infected with HIV and simian immunodeficiency virus (SIV) specific CD8 + T cells in the rhesus macaque model of HIV transmission and infection. We are particularly interested in using this method to identify immune correlates of protection from these viruses. With these objectives, we used MHC tetramers loaded with SIV Gag and Tat peptides to identify Gag and Tat specific CD8 + T cells in SIV infected macaques. We have successfully analyzed Gag and Tat specific CD8 + cells in lymph node, spleen, cervix and vagina tissues (unpublished data, and Mothe´ et al., 2002). An example of IST staining is shown in Fig. 3, where Gag specific T cells are evident in the submucosa of vaginal sections of chronically SIV infected macaques. In these studies of tissues from SIV infected macaques, Gag and Tat specific CD8 + T cells, when present, were abundant and widely dispersed throughout CD8 + T cell rich regions of every tissue examined. Intermixed within the tissues we observed cells that were stained intensely with tetramers and weakly with CD8 antibodies, as well as cells that were stained intensely with CD8 antibodies and weakly with tetramers. In addition, we occasionally found regions with clusters of a few to dozens of tetramer positive cells in close proximity to each other, possibly representing sites of clonal expansion. Daniels and Jameson (2000) showed that CD8 antibodies could interfere with or enhance tetramer binding to TCRs. In support of these findings we found that counter-staining tetramer stained cells with a clone of CD8 antibodies described as interfering by Daniels and Jameson yielded poorer quality tetramer staining compared to a clone of CD8 antibodies shown to enhance tetramer binding (Skinner et al., 2000). Therefore, we recommend that anti-CD8 antibodies be chosen carefully when counterstaining tetramer stained cells.
Fig. 3. Gag specific T cells in the submucosa of a vaginal section from a chronically SIV infected macaque. Fresh vaginal sections from 15 weeks post SIV infection were stained with (A) Mamu A * 01/Gag tetramers (red) and (B) CD8 antibodies (green). (C) Images from (A) and (B) merged. Thirteen confocal Z-scans were collected at 2-Am intervals using a 20 objective and merged.
In both Haanen et al. (2000) and Skinner et al. (2000), the punctate tetramer staining on the surface of T cells does not entirely overlap with CD8 staining (Fig. 1C). In addition, Haanan et al. observed increased punctate tetramer microclustering in cells incubated at 37 jC. These microclusters did not show significant colocalization with glycolipid-rich membrane domains (rafts) (Viola et al., 1999; Haanen et al., 2000). These findings were interpreted to suggest
P.J. Skinner, A.T. Haase / Journal of Immunological Methods 268 (2002) 29–34
that MHC binding the TCR during IST staining stimulates an early step in the formation of a TCR signaling site, but does not signify the formation of a mature signaling domain (Grakoui et al., 1999). Haanen et al. (2000) also examined influenza A specific CD8 + T cells in murine lung tissue after influenza infection and documented extensive infiltration of the pulmonary lobes with tetramer stained antigen specific CD8 + T cells. In a tumor rejection model, at the time of tumor rejection, they also visualized high concentrations of tumor specific T cells in draining lymph nodes as well as at the margins of the tumors. Within the tumor, many of the tetramer stained T cells abutted green fluorescent protein (GFP)-labeled tumor cells.
4. Prospects IST staining has opened the door to exploration of antigen specific T cells in tissues. As an example of the promise of IST staining, Masopust et al. (2001) showed that memory T cells localize predominantly to non-lymphoid tissues and that unlike memory CD8 + T cells in lymphoid compartments, memory CD8 + T cells in non-lymphoid tissues show effector lytic activity directly ex vivo. These findings indicate that CTLs in non-lymphoid tissues, unlike their lymphoid counterparts, are poised for an immediate attack in response to infection. These findings demonstrate the need to study CTLs in non-lymphoid tissues in addition to CTLs in lymph tissue in order to accurately characterize the antigen specific CTL response. Analyzing antigen specific CTLs in non-lymphoid tissues by flow cytometry is technically challenging as it can be difficult to extract sufficient numbers of T cells from tissues for analysis. IST staining overcomes this obstacle and provides an opportunity to investigate antigen specific CTLs at many sites. We can envision many other applications of IST staining of antigen specific T cells. Combined with staining with antibodies to activation markers, integrins, and other markers will lead to a greater understanding of how antigen specific CD8 + T cells migrate, differentiate and function. We are currently focusing on combining IST staining with in situ hybridization (ISH) to study the relationship of HIV and SIV specific CD8 + T cells
33
and HIV to SIV infected cells. This will allow us to not only visualize this relationship in various tissues at various times of infection, but also allow us to quantify target to effector ratios within tissues. The use of IST staining combined with ISH will also allow us to determine the relationship of productively infected cells and virus specific CD8 + T cells compared to the spatial relationship of non-productively infected cells and virus specific T cells. These studies will certainly lead to an increased understanding of antiviral immunity and protective correlates of natural infection or when the immune response has been enhanced, e.g. by vaccination or antiviral treatment interruptions. It may be possible also, with IST staining, to characterize the cellular biology of the T cell response in vivo by determining the subcellular localization of tetramer stained TCRs in relation to other cellular components. IST staining can be used to examine the punctate surface staining of TCRs in relation to other membrane components such as co-receptors and other signaling molecules and monitor how these relationships change during naı¨ve, activated, and memory states, and in response to certain stimuli. Perhaps the combination of real time video microscopy with IST staining could be used to monitor TCR interactions and internalization in responses to different stimuli. It will also be interesting to determine how relationship of TCRs and other cellular components changes at the subcellular level, as T cells migrate through different tissues. Thus, IST staining can be used not only for future studies of antigen specific T cells in tissues, but can also be utilized to broaden our understanding of TCR dynamics at the cellular and organ system level.
References Altman, J.D., Moss, P.A.H., Goulder, P.J.R., Barouch, D.H., McHeyzer-Williams, M.G., Bell, J.I., McMichael, A.J., Davis, M.M., 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94 – 96 [published erratum appears in Science 1998 Jun 19; 280(5371) 1821]. Daniels, M.A., Jameson, S.C., 2000. Critical role for CD8 in T cell receptor binding and activation by Peptide/Major histocompatibility complex multimers. J. Exp. Med. 191 (2), 335 – 346. Grakoui, A., Bromley, S.K., Sumen, C., Davis, M.M., Shaw, A.S., Allen, P.M., Dustin, M.L., 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221 – 227.
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Haanen, J.B., van Oijen, M.G., Tirion, F., Oomen, L.C., Kruisbeek, A.M., Vyth-Dreese, F.A., Schumacher, T.N., 2000. In situ detection of virus- and tumor-specific T-cell immunity. Nat. Med. 6, 1056 – 1060. Hogquist, K.A., Jameson, S.C., Heath, W.R., Howard, J.L., Bevan, M.J., Carbone, F.R., 1994. T cell receptor antagonist peptides induce positive selection. Cell 76, 17 – 27. Kearney, E.R., Pape, K.A., Loh, D.Y., Jenkins, M.K., 1994. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1, 327 – 339. Mamalaki, C., Elliott, J., Norton, T., Yannoutsos, N., Townsend, A.R., Chandler, P., Simpson, E., Kioussis, D., 1993. Positive and negative selection in transgenic mice expressing a T-cell receptor specific for influenza nucleoprotein and endogenous superantigen. Dev. Immunol. 3, 159 – 174. Masopust, D., Vezys, V., Marzo, A.L., Lefrancois, L., 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science 291 (5512), 2413 – 2417. Mothe´, B.R., Horton, H., Carter, D.K., Liebl, M.E., Skinner, P.J., Allen, T.M., Vogel, T.U., Franchini, G., Rehrauer, W., Watkins,
D.I., 2002. Dominance of CD8 responses specific for epitopes bound by a single MHC class I molecular during both the acute and chronic phases of viral infection. J. Virol. 76, 875 – 884. Schmidt, C.S., Mescher, M.F., 1999. Adjuvant effect of IL-12: conversion of peptide antigen administration from tolerizing to immunizing for CD8+ T cells in vivo. J. Immunol. 163, 2561 – 2567. Sha, W.C., Nelson, C.A., Newberry, R.D., Kranz, D.M., Russell, J.H., Loh, D.Y., 1988. Selective expression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice. Nature 335, 271 – 274. Skinner, P.J., Daniels, M.A., Schmidt, C.S., Jameson, S.C., Haase, A.T., 2000. Cutting edge: in situ tetramer staining of antigenspecific T cells in tissues. J. Immunol. 165, 613 – 617. Toes, R.E., Offringa, R., Blom, R.J., Melief, C.J., Kast, W.M., 1996. Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction. Proc. Natl. Acad. Sci. U. S. A. 93, 7855 – 7860. Viola, A., Schroeder, S., Sakakibara, Y., Lanzavecchia, A., 1999. T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science 283, 680 – 682.
Review
In situ tetramer staining Pamela J. Skinner, Ashley T. Haase * Department of Microbiology and Great Lakes Center for AIDS Research, University of Minnesota Medical School, MMC 196, 420 Delaware St. SE, Minneapolis, MN 55455, USA Received 1 October 2001; accepted 14 January 2002
Abstract The development of MHC tetramer staining has opened the doors to multiple avenues of new research [Science 274 (1996) 94]. In this review, we will discuss the development and application of in situ MHC tetramer (IST) staining. We describe two independently developed IST staining methodologies and discuss current uses, limitations, future uses and the interesting biology revealed by the use of IST staining. D 2002 Elsevier Science B.V. All rights reserved. Keywords: In situ tetramer staining; Vibratome; HIV; SIV
1. Development of the technique 1.1. Indirect IST staining The development of MHC tetramer staining has revolutionized our ability to study antigen specific T cells (Altman et al., 1996), but until recently, its use has been limited to ex vivo studies. We sought to expand this methodology and describe here the successful development of a technique that extends tetramer staining to visualize antigen specific CD8 + T cells in tissues with their spatial relationship to other cells intact. In order to expedite the development of an in
Abbreviations: IST staining, in situ tetramer staining; TCR, T cell receptor; HIV, human immunodeficiency virus; SIV, simian immunodeficiency virus; ISH, in situ hybridization; APC, allophycocyanin; GFP, green fluorescent protein. * Corresponding author. Tel.: +1-612-624-4442; fax: +1-612626-0623. E-mail address: [email protected] (A.T. Haase).
situ tetramer (IST) staining technique, we utilized T cell receptor (TCR) transgenic mice (Sha et al., 1988; Hogquist et al., 1994). In these mice, the vast majority of T cells are identical. Our strategy involved making MHC class I tetramers with ExtraAvidin-FITC (Sigma) and then using FITC as an epitope to amplify the tetramer signal. Because we thought that it would be important to maintain the mobility of TCRs to interact with tetramers, we initiated studies using fresh unfixed tissue sections. Using a vibratome (TPI), we generated 200 Am thick fresh spleen sections. The sections were stained in a solution containing 0.5 Ag/ml tetramers, CD8 antibodies, 2% normal goat serum and PBS and incubated at 4jC overnight. The sections were then fixed, and the signal from the tetramers was amplified by incubating the sections with rabbit anti-FITC antibodies followed by incubation with goat-anti-rabbit conjugated to Cy3 (or other fluorophores). This strategy was successful and an example of these results is shown in Fig. 1 (Skinner et al., 2000). As negative controls, we either stained sections with MHC tet-
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 1 9 7 - 7
30
P.J. Skinner, A.T. Haase / Journal of Immunological Methods 268 (2002) 29–34
Fig. 1. MHC tetramers used to stain fresh spleen sections from 2C transgenic mice. (A – C) A section that was stained with Kb/SIY tetramers (A, red) and anti-CD8 clone 53-6.7 antibodies (B, green). (C) Images from (A) and (B) merged. (D – F) A section that was stained with negative control Kb/OVA tetramers (D, red) and anti-CD8 clone 53-6.7 (E, green). (F) Images from (D) and (E) merged. The confocal Z scans shown in (A) and (D) were collected using the same parameters with a 60 objective. The spleen used in this experiment was stored in PBS at 4j for 24 h prior to sectioning and staining. Reprinted with permission from Skinner et al. (2000). Copyright 2000. The American Association of Immunologists.
ramers loaded with an irrelevant peptide or we stained spleen sections from a different line of TCR transgenic mice. We then tested the method in a more realistic system where only a small fraction of the CD8 + T cell population is antigen specific. We analyzed spleen sections from a wild type mouse in which transgenic T cells had been adoptively transferred (Kearney et al., 1994; Schmidt and Mescher, 1999) (Fig. 2). Tetramer reactive CD8 + T cells were counted and the percentage of CD8 + cells that were tetramer positive was determined. Cells from half of the same spleen were analyzed by flow cytometry and the percentage of CD8 + T cells stained with tetramers was determined. Both the tetramer stained sections and the flow cytometry data showed that f 1% of the CD8 + T cells stained with tetramers. Thus, IST staining is equivalent in specificity and in sensitivity to flow cytometry.
In order to analyze archived tissue samples, we next determined whether IST staining would work with fixed or frozen tissues. Using spleens from TCR transgenic mice (Sha et al., 1988; Hogquist et al., 1994), we could detect tetramer staining in spleen tissue that had been lightly fixed in 2% formaldehyde for 20 min, or fixed in 50% methanol and 50% acetone for 5 min at 20 jC. However, the intensity of staining was much weaker and the background higher than unfixed fresh tissue sections. Antigen specific CD8 + T cells were also detected in frozen tissue sections. Frozen sections, 10 Am thick, on slides, were kept wet throughout the staining procedure, and during washing steps many sections were lost. The remaining stained sections showed tetramer positive cells, but the staining was not nearly as intense compared to fresh tissue. Moreover, in principle 20 times as much tissue can be examined for antigen specific CD8 + T cells in thick fresh sections.
P.J. Skinner, A.T. Haase / Journal of Immunological Methods 268 (2002) 29–34
31
been described in (Haanen et al., 2000). They also used fresh spleen sections from TCR transgenic mice and sections from spleens with adoptively transferred antigen specific T cells (Mamalaki et al., 1993; Toes et al., 1996) and exploited the high intensity signal emitted in MHC class I tetramers labeled with allophycocyanin (APC) to directly stain antigen specific T cells. Tetramer stained cells in the tissue sections were quantitated and compared to quantities obtained by flow cytometry and found to be similar. Comparison of the two described IST staining methods showed advantages and disadvantages for each. The intensity of staining using the indirect method, with amplification, is greater than the direct staining method, measured by the confocal laser power needed for detection (maximum confocal laser power used to see APC labeled tetramers, P. Skinner, unpublished). Because of the weaker signal, we were not able to sample as deeply into the tissue. The major advantage we have found with the direct staining method is lower background staining and the ability to more easily colabel with antibodies to other interesting proteins in the tissue.
2. Limitations
Fig. 2. Adoptively transferred T cells detected in situ using MHC tetramers. Fresh spleen sections from a wild type mouse that 2C transgenic mouse lymphocytes were adoptively transferred were stained with (A) Kb/SIY tetramers (red) and (B) anti-CD8 antibodies (green). (C) Images from (A) and (B) merged. Reprinted with permission from Skinner et al. (2000). Copyright 2000. The American Association of Immunologists.
For these reasons, we have subsequently focused on and recommend using fresh tissues for IST staining. 1.2. Direct IST staining Another IST staining method using MHC class I tetramers to stain antigen specific CD8 + T cells has
IST staining is limited in the same ways that exvivo MHC tetramer techniques are limited. The technique requires cloned and characterized MHC molecules and MHC matched tissue. These are sometimes difficult to obtain. Another limitation is that individual MHC tetramers only detect a subset of the total pool of antigen specific T cells directed against specific pathogens. Given that an individual can have up to six different MHC class I alleles, and pathogens are comprised of innumerable immunogenic peptides, using MHC tetramers to study the entire repertoire of CTL response to a pathogen is daunting. However, increasing numbers of MHC alleles from multiple species are rapidly being cloned and characterized making broad studies of CTLs more feasible. An additional limitation with IST staining is the requirement of fresh tissue specimens for optimal results. The requirement for fresh tissues will likely also require, in the short term, new prospective studies. Fresh tissues must be processed immediately. However, they can be obtained at a distant site and shipped on ice and
32
P.J. Skinner, A.T. Haase / Journal of Immunological Methods 268 (2002) 29–34
sectioned and stained the following day without significant loss of signal.
3. Summary of current practices and examples of interesting biology revealed Our primary objective in developing an IST staining technique was to use IST staining to learn more about human immunodeficiency virus (HIV) specific CD8 + T cells in humans infected with HIV and simian immunodeficiency virus (SIV) specific CD8 + T cells in the rhesus macaque model of HIV transmission and infection. We are particularly interested in using this method to identify immune correlates of protection from these viruses. With these objectives, we used MHC tetramers loaded with SIV Gag and Tat peptides to identify Gag and Tat specific CD8 + T cells in SIV infected macaques. We have successfully analyzed Gag and Tat specific CD8 + cells in lymph node, spleen, cervix and vagina tissues (unpublished data, and Mothe´ et al., 2002). An example of IST staining is shown in Fig. 3, where Gag specific T cells are evident in the submucosa of vaginal sections of chronically SIV infected macaques. In these studies of tissues from SIV infected macaques, Gag and Tat specific CD8 + T cells, when present, were abundant and widely dispersed throughout CD8 + T cell rich regions of every tissue examined. Intermixed within the tissues we observed cells that were stained intensely with tetramers and weakly with CD8 antibodies, as well as cells that were stained intensely with CD8 antibodies and weakly with tetramers. In addition, we occasionally found regions with clusters of a few to dozens of tetramer positive cells in close proximity to each other, possibly representing sites of clonal expansion. Daniels and Jameson (2000) showed that CD8 antibodies could interfere with or enhance tetramer binding to TCRs. In support of these findings we found that counter-staining tetramer stained cells with a clone of CD8 antibodies described as interfering by Daniels and Jameson yielded poorer quality tetramer staining compared to a clone of CD8 antibodies shown to enhance tetramer binding (Skinner et al., 2000). Therefore, we recommend that anti-CD8 antibodies be chosen carefully when counterstaining tetramer stained cells.
Fig. 3. Gag specific T cells in the submucosa of a vaginal section from a chronically SIV infected macaque. Fresh vaginal sections from 15 weeks post SIV infection were stained with (A) Mamu A * 01/Gag tetramers (red) and (B) CD8 antibodies (green). (C) Images from (A) and (B) merged. Thirteen confocal Z-scans were collected at 2-Am intervals using a 20 objective and merged.
In both Haanen et al. (2000) and Skinner et al. (2000), the punctate tetramer staining on the surface of T cells does not entirely overlap with CD8 staining (Fig. 1C). In addition, Haanan et al. observed increased punctate tetramer microclustering in cells incubated at 37 jC. These microclusters did not show significant colocalization with glycolipid-rich membrane domains (rafts) (Viola et al., 1999; Haanen et al., 2000). These findings were interpreted to suggest
P.J. Skinner, A.T. Haase / Journal of Immunological Methods 268 (2002) 29–34
that MHC binding the TCR during IST staining stimulates an early step in the formation of a TCR signaling site, but does not signify the formation of a mature signaling domain (Grakoui et al., 1999). Haanen et al. (2000) also examined influenza A specific CD8 + T cells in murine lung tissue after influenza infection and documented extensive infiltration of the pulmonary lobes with tetramer stained antigen specific CD8 + T cells. In a tumor rejection model, at the time of tumor rejection, they also visualized high concentrations of tumor specific T cells in draining lymph nodes as well as at the margins of the tumors. Within the tumor, many of the tetramer stained T cells abutted green fluorescent protein (GFP)-labeled tumor cells.
4. Prospects IST staining has opened the door to exploration of antigen specific T cells in tissues. As an example of the promise of IST staining, Masopust et al. (2001) showed that memory T cells localize predominantly to non-lymphoid tissues and that unlike memory CD8 + T cells in lymphoid compartments, memory CD8 + T cells in non-lymphoid tissues show effector lytic activity directly ex vivo. These findings indicate that CTLs in non-lymphoid tissues, unlike their lymphoid counterparts, are poised for an immediate attack in response to infection. These findings demonstrate the need to study CTLs in non-lymphoid tissues in addition to CTLs in lymph tissue in order to accurately characterize the antigen specific CTL response. Analyzing antigen specific CTLs in non-lymphoid tissues by flow cytometry is technically challenging as it can be difficult to extract sufficient numbers of T cells from tissues for analysis. IST staining overcomes this obstacle and provides an opportunity to investigate antigen specific CTLs at many sites. We can envision many other applications of IST staining of antigen specific T cells. Combined with staining with antibodies to activation markers, integrins, and other markers will lead to a greater understanding of how antigen specific CD8 + T cells migrate, differentiate and function. We are currently focusing on combining IST staining with in situ hybridization (ISH) to study the relationship of HIV and SIV specific CD8 + T cells
33
and HIV to SIV infected cells. This will allow us to not only visualize this relationship in various tissues at various times of infection, but also allow us to quantify target to effector ratios within tissues. The use of IST staining combined with ISH will also allow us to determine the relationship of productively infected cells and virus specific CD8 + T cells compared to the spatial relationship of non-productively infected cells and virus specific T cells. These studies will certainly lead to an increased understanding of antiviral immunity and protective correlates of natural infection or when the immune response has been enhanced, e.g. by vaccination or antiviral treatment interruptions. It may be possible also, with IST staining, to characterize the cellular biology of the T cell response in vivo by determining the subcellular localization of tetramer stained TCRs in relation to other cellular components. IST staining can be used to examine the punctate surface staining of TCRs in relation to other membrane components such as co-receptors and other signaling molecules and monitor how these relationships change during naı¨ve, activated, and memory states, and in response to certain stimuli. Perhaps the combination of real time video microscopy with IST staining could be used to monitor TCR interactions and internalization in responses to different stimuli. It will also be interesting to determine how relationship of TCRs and other cellular components changes at the subcellular level, as T cells migrate through different tissues. Thus, IST staining can be used not only for future studies of antigen specific T cells in tissues, but can also be utilized to broaden our understanding of TCR dynamics at the cellular and organ system level.
References Altman, J.D., Moss, P.A.H., Goulder, P.J.R., Barouch, D.H., McHeyzer-Williams, M.G., Bell, J.I., McMichael, A.J., Davis, M.M., 1996. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94 – 96 [published erratum appears in Science 1998 Jun 19; 280(5371) 1821]. Daniels, M.A., Jameson, S.C., 2000. Critical role for CD8 in T cell receptor binding and activation by Peptide/Major histocompatibility complex multimers. J. Exp. Med. 191 (2), 335 – 346. Grakoui, A., Bromley, S.K., Sumen, C., Davis, M.M., Shaw, A.S., Allen, P.M., Dustin, M.L., 1999. The immunological synapse: a molecular machine controlling T cell activation. Science 285, 221 – 227.
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