Cutting Edge: In Situ Tetramer Staining of
Antigen-Specific T Cells in Tissues
This information is current as
of June 17, 2017.
Subscription
Permissions
Email Alerts
J Immunol 2000; 165:613-617; ;
doi: 10.4049/jimmunol.165.2.613
http://www.jimmunol.org/content/165/2/613
This article cites 22 articles, 16 of which you can access for free at:
http://www.jimmunol.org/content/165/2/613.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
References
Pamela J. Skinner, Mark A. Daniels, Clint S. Schmidt,
Stephen C. Jameson and Ashley T. Haase
●
Cutting Edge: In Situ Tetramer
Staining of Antigen-Specific T Cells in
Tissues1
Pamela J. Skinner,* Mark A. Daniels,†
Clint S. Schmidt,† Stephen C. Jameson,† and
Ashley T. Haase*2
T
he introduction by Altman et al. of a method to identify
and phenotypically characterize Ag-specific T lymphocytes has quickly advanced understanding of the immune
response in general and particularly the virus-specific CTL response to a number of acute and chronic infections that include
influenza virus, lymphocytic choriomeningitis virus, EBV, human
T cell leukemia virus-1, hepatitis C, Listeria monocytogenes, and
HIV-1 and SIV infections (1–11). Enumerating virus-specific
CD8⫹ T lymphocytes in cross-sectional or longitudinal studies has
made it possible to track expansion and contraction of the CTL
response in these infections and, in HIV-1 infection, to document
the inverse correlation between the CTL response and viral load
and progression to disease (4). Thus far, however, these studies
*Department of Microbiology and Great Lakes Center for AIDS Research, and †Center for Immunology and Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis, MN 55455
Received for publication February 14, 2000. Accepted for publication April 24, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Great Lakes Regional Center for AIDS Research
Grant AI 42586, National Institutes of Health Infectious Disease Training Grant AI
07421 (to P.J.S.), and National Institutes of Health Grant AI 28246.
2
Address correspondence and reprint requests to Dr. Ashley T. Haase, Department of
Microbiology, University of Minnesota Medical School, Box 196 Mayo, 420 Delaware Street SE, Minneapolis MN, 55455. E-mail address: [email protected]
Copyright © 2000 by The American Association of Immunologists
●
have been directed to cells isolated from blood, semen, or tissues
that were stained with tetramer-peptide complexes and then analyzed by flow cytometry. With the eventual goal of investigating
the spatial and temporal relationships between viral replication and
the specific CTL response in tissue, we sought to adapt the tetramer staining technology to tissue analysis. To that end, we set
out to find conditions in an optimal well-characterized transgenic
model for directly staining Ag-specific T cells in tissues. In this
report, we describe such a technique that should be generally applicable to visualizing Ag-specific T cells in tissues.
Materials and Methods
Generation of MHC tetramers/multimers
Biotinylated Kb/␤2-microglobulin/peptide molecules were generated with
either OVA (SIINFEKL) or SIY (SIYRYYGL) peptides as previously described (1, 12). Tetramers/multimers were generated by adding six aliquots
of FITC-labeled ExtraAvidin (Sigma, St. Louis, MO) over the course of 8 h
to either Kb/␤2-microglobulin/SIY or Kb/␤2-microglobulin/OVA to a final
molar ratio of 4.5:1.
Generation of spleen sections
2C (13) or OT-I (14) TCR transgenic mice on a C57BL/6 background or
wild-type C57BL/6 mice were used in these experiments. The adoptive
transfer of 2C transgenic cells into a C57BL/6 recipient was performed as
described (15). Fresh spleens or spleens stored overnight in PBS at 4°C
were cut into three pieces and embedded in 4% low-melt agarose, patted
dry, and secured to vibratome blocks with Loctite vibratome tissue adhesive (Ted Pella, Redding, CA). After letting the glue set for at least 3 min,
the blocks were placed in a vibratome bath containing 0°C PBS. A Vibratome 3000 (Technical Products International, St. Louis, MO) was used
to cut the tissue (16, 17). Vibratome sections, 200 ␮m thick, were generated with a dead slow speed and maximum amplification using a standard
double-edged razor blade set at an angle of 27°.
Detection of Ag-specific T cells in situ
Fresh sections were stained free floating in 1 ml solution with four sections
per well in 24-well tissue culture plates, and incubations were conducted at
4°C on a rocking platform. Tetramers were added at a concentration of 0.5
␮g/ml with 2% normal goat serum (NGS)3 and 0.5 ␮g/ml rat anti-CD8a
Abs clone 53-6.7 (PharMingen, San Diego, CA) or CTCD8a (Caltag, South
San Francisco, CA) and incubated overnight. Sections were washed with
PBS and then fixed with PBS-buffered 2% formaldehyde for 30 min at
room temperature. Sections were again washed in PBS and then incubated
with rabbit anti-FITC Abs (BioDesign, New York, NY or Zymed, San
Francisco, CA) diluted 1:10,000 in PBS with 2% NGS and incubated overnight. Sections were washed three times with PBS for at least 20 min and
then incubated with Cy3-conjugated goat anti-rabbit Abs and Cy5-conjugated goat anti-rat Abs (Jackson ImmunoResearch, West Grove, PA) both
3
Abbreviations used in this paper: NGS, normal goat serum; IST, in situ tetramer.
0022-1767/00/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Staining Ag-specific T cells with fluorescently labeled tetrameric MHC/peptide complexes has provided a powerful experimental approach to characterizing the immune response.
In this report, we describe an extension of this method to directly visualize Ag-specific T cells in tissues. We successfully
stained transgenic T cells with MHC tetramers in spleen sections from both 2C and OT-1 TCR transgenic mice. In addition, with the in situ tetramer staining technique, we detected
a very small population of Ag-specific T cells in tissue after
adoptive transfer of transgenic TCR T cells to a syngeneic nontransgenic mouse. We also show that the in situ tetramer technique can be applied to lightly fixed as well as frozen tissue,
thus extending the method to archived tissue collections. This
in situ tetramer staining technique offers a general approach to
tracking the Ag-specific T cells in tissues. The Journal of Immunology, 2000, 165: 613– 617.
614
CUTTING EDGE
diluted 1:1000 in PBS with 2% NGS overnight. Finally, sections were
washed three times for at least 20 min and then mounted to slides with
warmed glycerol gelatin (Sigma) containing 4 mg/ml n-propyl galate.
Stained sections were analyzed using a Bio-Rad 1000 confocal microscope
(Richmond, CA).
In some experiments, spleens were fixed in 2% formaldehyde buffered
with PBS for 30 min at room temperature before sectioning and staining.
The prefixed sections were stained as described for fresh sections with the
exception that they were not fixed after the tetramer incubation. Frozen
tissue sections, 10 ␮m thick, were also generated from spleen tissue that
had been frozen in OCT freezing medium (Tissue-Tek, OT Embedding
Medium, Sakura Finetek, Torrance, CA). These sections were stained as
described for fresh sections with the exception that sections were mounted
onto silane-coated slides before staining, incubations were performed with
200 ␮l of solution, PBS wash times were reduced to 5 min, and secondary
incubations were reduced to 3 h.
Results and Discussion
We chose 2C TCR transgenic mice (13) to initiate our attempts to
develop a method using MHC class I tetramers to identify Agspecific T cells in situ because 2C receptors bind several ligands
including the Kb/SIY molecule with relatively high affinity and
Kb/SIY tetramers have been used to identify 2C T cells ex vivo
(12, 18, 19). Most T cells in 2C transgenic mice are CD8⫹ but
there are some CD8⫺ 2C⫹ T cells (13, 20) (data not shown). We
conjugated biotinylated Kb/SIY monomers with FITC-labeled
ExtraAvidin to make Kb/SIY tetramers. Because tetramer staining
has thus far only been successfully applied to viable cells, we
initiated our studies in tissues sectioned with a vibratome (16, 17).
Using the Kb/SIY tetramers, we stained 200-␮m fresh spleen sections from 2C transgenic mice. To detect bound tetramers, we
amplified the signal using purified rabbit Abs directed against
FITC followed by anti-rabbit Abs conjugated to Cy3 (Fig. 1, A and
C). Sections were counterstained with anti-CD8 Abs as a positive
control (Fig. 1, B and C). The merged image of Kb/SIY staining
and anti-CD8 staining (Fig. 1C) shows that the tetramer-positive
cells counterstained with anti-CD8 Abs. Without amplification, no
tetramer signal was detected (not shown). As a negative control,
spleen sections from 2C transgenic mice were stained with tetramers of Kb molecules loaded with an irrelevant peptide SIINFEKL
from OVA (Fig. 1, D and F) and counterstained with anti-CD8
Abs (Fig. 1, E and F). In contrast to the Kb/SIY tetramers, the
Kb/OVA tetramers did not stain the CD8⫹ 2C transgenic T cells
(Fig. 1, D–F). As an additional negative control, spleen sections
from wild-type C57BL/6 were stained with Kb/SIY tetramers, and
these did not show tetramer-specific staining (not shown). However, the anti-FITC/anti-rabbit-Cy3 Abs, used to amplify the tetramer signal, did label a small subset of cells that were not further
evaluated because they typically were outside of the T cell-rich
white pulp of the spleen, where they would not be confused with
tetramer-positive T cells. Because this subset was also CD8⫺, in
all experiments sections were counterstained with anti-CD8 Abs so
that doubly stained cells that bound the labeled peptide/MHC complex could be unequivocally distinguished from background staining. Using a confocal microscope, tetramer-stained cells could be
detected as far as 120 ␮m into the tissue. This technique has considerable analytical and sampling power because MHC class I tetramers
can stain Ag-specific T cells in situ through this depth of tissue.
We subsequently investigated whether in situ tetramer (IST)
staining would work using a different system. We used Kb/OVA
tetramers to stain OVA-specific T cells from OT-I transgenic mice
(14) (Fig. 2A). As a negative control, parallel sections were stained
with Kb/SIY tetramers and did not show tetramer staining (Fig.
2B). Thus, the IST staining technique was used successfully to
stain Ag-specific T cells in tissues from 2C as well as OT-I
transgenic mice.
To see if we could use MHC tetramers to detect Ag-specific T cells
in a system in which only a fraction of a percentage of the T cells are
transgenic, we adoptively transferred lymphocytes from 2C transgenic mice into a wild-type C57BL/6 mouse (21, 22). Five days after
the adoptive transfer, spleen and lymph tissue was collected. Flow
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 1. MHC tetramers used to stain fresh spleen sections from 2C transgenic mice. A–C show a section that was stained with Kb/SIY tetramers
(A, red) and anti-CD8 clone 53-6.7 Abs (B, green); C shows the images from A and B merged. D–F show a section that was stained with Kb/OVA multimers
(D, red) and anti-CD8 clone 53-6.7 (E, green); F shows the images from D and E merged. The images shown in A and D were collected using the same
confocal parameters. The spleen used in this experiment was stored in PBS at 4°C for 24 h before sectioning and staining.
The Journal of Immunology
615
cytometric analysis revealed that the transferred transgenic T cells
comprised 0.2– 0.4% of the total lymphocytes (not shown). Spleen
sections stained with Kb/SIY tetramers (Fig. 3, A and C) and anti-CD8
Abs (Fig. 3, B and C) showed staining of the 2C transgenic T cells.
The merged image of Fig. 3, A and B shows that the Kb/SIY tetramerstained cells were CD8⫹ (Fig. 3C). In contrast, control sections from
a mouse in which no 2C cells were transferred were stained with
Kb/SIY and did not show tetramer staining (not shown). Quantitation
revealed that 23 of 2306 or 1.0% of the CD8⫹ T cells were stained
with Kb/SIY tetramers in the adoptively transferred spleen. In contrast, 1 of 2742 or 0.04% of the CD8⫹ T cells were stained with
Kb/SIY in the control sections. To corroborate these findings, flow
cytometry was used to calculate the percentage of transgenic T cells
from a portion of the same spleen presented in Fig. 3. Flow cytometry
identified a comparable stained population of 1.3% of CD8⫹ T cells
(not shown). Thus, these data demonstrate that IST staining can be
used to stain small populations of Ag-specific T cells at a sensitivity
that is comparable to flow cytometry.
Recently, Daniels et al. reported that anti-CD8 Abs, depending
on the clone, either interfere with or enhance tetramer binding its
TCR ligand (12). Clone CTCD8a was shown to inhibit Kb/SIY
tetramer binding. In contrast, clone 53-6.7 was shown to enhance
tetramer binding. To assess this effect in the IST staining technique, we stained spleen sections from 2C transgenic mice with
Kb/SIY tetramers and the anti-CD8 clone CTCD8a at a concentration of 0.5 ␮g/ml. We found that anti-CD8 clone CTCD8a had
a negative effect on tetramer staining (not shown). Given that some
anti-CD8 Abs can interfere with tetramer binding, care should be
taken when selecting a CD8 Ab to counterstain cells, and minimal
amounts of anti-CD8 should be used.
Frequently, investigations involve collaborations in which tissue
is collected at one institution and analyzed at another. Also, investigations sometimes involve limited amounts of tissue and/or
tissue that has been frozen and archived. To facilitate the investigation of Ag-specific T cells in these circumstances, we tested
whether IST staining could be performed on tissue that was either
FIGURE 3. Adoptively transferred T cells detected in situ using MHC tetramers. T cells from a 2C transgenic mouse were adoptively transferred into
a wild-type mouse. After euthanizing this mouse, fresh spleen sections were generated and stained with Kb/SIY tetramers (A, red) and anti-CD8 Abs
(B, green). C shows the images from A and B merged.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 2. MHC tetramers used to stain fresh spleen sections from OT-1 transgenic mice. A–C shows a section that was stained with Kb/OVA tetramers
(A, red) and anti-CD8 (B, green); C shows the images from A and B merged. D–F shows a parallel section that was stained with Kb/SIY tetramers (D, red)
and anti-CD8 (E, green); F shows the images from D and E merged. The same confocal parameters were used to capture both images.
616
CUTTING EDGE
stored overnight in PBS at 4°C, lightly fixed with 2% formaldehyde, lightly fixed with 50% acetone and 50% methanol, or frozen.
Spleens from 2C transgenic mice were either stored at 4°C in PBS
overnight before sectioning and staining or sectioned and stained
shortly after dissection. The sections from spleens that were stored
overnight showed identical staining as the spleens that were sectioned and stained promptly after dissection. An example of tissue
that was stored overnight before sectioning and staining is shown
in Fig. 1. We were also successful in staining sections from spleens
prefixed for 30 min in 2% formaldehyde (Fig. 4) or prefixed in
50% acetone and 50% methanol at ⫺20°C for 5 min (not shown)
that were stored in PBS overnight at 4°C prior to sectioning and
staining. Finally, we were also successful in staining 2C transgenic
T cells in 10-␮m thick frozen sections from spleens from 2C transgenic mice with Kb/SIY tetramers (Fig. 5). The best quality staining with regard to cellular morphology was achieved with tissue
that was not fixed or frozen before staining. In addition, the
200-␮m thick sections offered more information than the 10-␮m
frozen sections as about 10 times more cells could be analyzed per
section. Accordingly, when examining frozen tissue from a system
with relatively low levels of Ag-specific T cells in situ, thick sec-
tions should be generated to increase the number of positive cells
per section.
In summary, we have developed a technique to stain Ag-specific
T cells in situ. We have successfully used MHC class I tetramers
to stain Ag-specific T cells from 2C and OT-1 transgenic mice, as
well as 2C transgenic T cells that were adoptively transferred into
a wild-type mouse. We point out that careful selection of anti-CD8
Abs is important when colabeling with tetramers, as anti-CD8 Abs
can alter tetramer binding in situ. Finally, we demonstrated that
our technique can be used to stain Ag-specific T cells in tissue that
has been stored overnight, lightly fixed, or frozen. We think this
technique, the first to be described that allows the detection of
Ag-specific T cells in tissues, promises to be a valuable tool for
studies of Ag-specific T cells in vivo.
Acknowledgments
We thank Marc Jenkins for valuable discussions regarding this work, Matt
Mescher for reviewing the manuscript and providing resources used to
generate these data, Debra Lins for technical assistance, and Linda Boland,
Grant Yeaman, and staff at Technical Products International for helpful
advice on generating fresh tissue sections.
FIGURE 5. MHC tetramer-stained frozen tissue sections. Frozen sections were generated from 2C transgenic mouse spleens and stained with either
Kb/SIY multimers (A) or Kb/OVA multimers (B). Images were captured using the same confocal microscope parameters.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 4. MHC tetramers used to stain lightly fixed tissue. Spleens from 2C transgenic mice were fixed for 30 min in 2% formaldehyde and stored
for 24 h in PBS at 4°C before sectioning and staining. Parallel sections were stained with either Kb/SIY tetramers (A) or Kb/OVA tetramers (B). The same
confocal parameters were used to capture these images.
The Journal of Immunology
617
References
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
1. Altman, J. D., P. A. H. Moss, P. J. R. Goulder, D. H. Barouch,
M. G. McHeyzer-Williams, J. I. Bell, A. J. McMichael, and M. M. Davis. 1996.
Phenotypic analysis of antigen-specific T lymphocytes. [Published erratum appears in 1998 Science 280:1821.] Science 274:94.
2. Doherty, P. C. 1998. The numbers game for virus-specific CD8⫹ T cells. Science
280:227.
3. McMichael, A. J., and C. A. O’Callaghan. 1998. A new look at T cells. J. Exp.
Med. 187:1367.
4. Ogg, G. S., X. Jin, S. Bonhoeffer, P. R. Dunbar, M. A. Nowak, S. Monard,
J. P. Segal, Y. Cao, S. L. Rowland-Jones, V. Cerundolo, et al. 1998. Quantitation
of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279:2103.
5. Busch, D. H., I. M. Pilip, S. Vijh, and E. G. Pamer. 1998. Coordinate regulation
of complex T cell populations responding to bacterial infection. Immunity 8:353.
6. Kuroda, M. J., J. E. Schmitz, D. H. Barouch, A. Craiu, T. M. Allen, A. Sette,
D. I. Watkins, M. A. Forman, and N. L. Letvin. 1998. Analysis of Gag-specific
cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class
I-peptide complex. J. Exp. Med. 187:1373.
7. Kuroda, M. J., J. E. Schmitz, W. A. Charini, C. E. Nickerson, C. I. Lord,
M. A. Forman, and N. L. Letvin. 1999. Comparative analysis of cytotoxic T
lymphocytes in lymph nodes and peripheral blood of simian immunodeficiency
virus-infected rhesus monkeys. J. Virol. 73:1573.
8. Jordan, H. L., M. J. Kuroda, J. E. Schmitz, T. Steenbeke, M. A. Forman, and
N. L. Letvin. 1999. Detection of simian immunodeficiency virus Gag-specific
CD8⫹ T lymphocytes in semen of chronically infected rhesus monkeys by cell
staining with a tetrameric major histocompatibility complex class I-peptide complex. J. Virol. 73:4508.
9. Egan, M. A., M. J. Kuroda, G. Voss, J. E. Schmitz, W. A. Charini, C. I. Lord,
M. A. Forman, and N. L. Letvin. 1999. Use of major histocompatibility complex
class I/peptide/␤2m tetramers to quantitate CD8⫹ cytotoxic T lymphocytes specific for dominant and nondominant viral epitopes in simian-human immunodeficiency virus-infected rhesus monkeys. J. Virol. 73:5466.
10. Greten, T. F., J. E. Slansky, R. Kubota, S. S. Soldan, E. M. Jaffee, T. P. Leist,
D. M. Pardoll, S. Jacobson, and J. P. Schneck. 1998. Direct visualization of
antigen-specific T cells: HTLV-1 Tax11–19-specific CD8⫹ T cells are activated
in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients. Proc. Natl. Acad. Sci. USA 95:7568.
He, X. S., B. Rehermann, F. X. Lopez-Labrador, J. Boisvert, R. Cheung,
J. Mumm, H. Wedemeyer, M. Berenguer, T. L. Wright, M. M. Davis, and
H. B. Greenberg. 1999. Quantitative analysis of hepatitis C virus-specific CD8⫹
T cells in peripheral blood and liver using peptide-MHC tetramers. Proc. Natl.
Acad. Sci. USA 96:5692.
Daniels, M. A., and S. C. Jameson. 2000. Critical role for CD8 in T cell receptor
binding and activation by Peptide/Major histocompatibility complex multimers.
J. Exp. Med. 191:335.
Sha, W. C., C. A. Nelson, R. D. Newberry, D. M. Kranz, J. H. Russell, and
D. Y. Loh. 1988. Selective expression of an antigen receptor on CD8-bearing T
lymphocytes in transgenic mice. Nature 335:271.
Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, and
F. R. Carbone. 1994. T cell receptor antagonist peptides induce positive selection.
Cell 76:17.
Schmidt, C. S., and M. F. Mescher. 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.
Smith, R. E. 1970. Comparative evaluation of 2 instruments and procedures to cut
nonfrozen sections. J. Histochem. Cytochem. 18:590.
Gonzalez, M., F. Mackay, J. L. Browning, M. H. Kosco-Vilbois, and R. J. Noelle.
1998. The sequential role of lymphotoxin and B cells in the development of
splenic follicles. J. Exp. Med. 187:997.
Udaka, K., K. H. Wiesmuller, S. Kienle, G. Jung, and P. Walden. 1996. SelfMHC-restricted peptides recognized by an alloreactive T lymphocyte clone.
J. Immunol. 157:670.
Sykulev, Y., Y. Vugmeyster, A. Brunmark, H. L. Ploegh, and H. N. Eisen. 1998.
Peptide antagonism and T cell receptor interactions with peptide-MHC complexes. Immunity 9:475.
Cai, Z., and J. Sprent. 1994. Resting and activated T cells display different requirements for CD8 molecules. J. Exp. Med. 179:2005.
Kearney, E. R., K. A. Pape, D. Y. Loh, and M. K. Jenkins. 1994. Visualization
of peptide-specific T cell immunity and peripheral tolerance induction in vivo.
Immunity 1:327.
Kedl, R. M., and M. F. Mescher. 1997. Migration and activation of antigenspecific CD8⫹ T cells upon in vivo stimulation with allogeneic tumor. J. Immunol. 159:650.