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* Center for Neurologic Diseases, Brigham and Women’s Hospital, and
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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8.0) and neuraminidase treatment enhances the staining capacity of
PLP139–151 tetramer without compromising specificity.
Furthermore, we found that induction of calcium fluxing by
tetramers in T cells may be used as a sensitive measure to detect
autoreactive T cells with a low affinity. Taken together, the data show
that the tetrameric reagent binds and stimulates
PLP139–151-reactive T cells with specificity. This
tetrameric reagent will be useful in studying the evolution of
PLP139–151-specific repertoire in naive mice and its
expansion during the autoimmune disease experimental autoimmune
encephalomyelitis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We and others have identified proteolipid protein (PLP)3139–151 and PLP178–191 as the major immunogenic and encephalitogenic epitopes that induce murine experimental autoimmune encephalomyelitis in the SJL mouse strain (13, 14, 15, 16). Although these epitopes of PLP bind MHC II IAs with similar affinities, the immune response to PLP139–151 is always dominant (16). Recently, based on limiting dilution analysis, we reported that naive SJL mice have a high frequency (1/20,000) of PLP139–151-reactive CD4 T cells circulating in the periphery (17). It is very clear that limiting dilution analysis does not give a true estimate of Ag-reactive cells in the repertoire, and to be able to determine more precisely the frequency of PLP139–151-reactive cells in the naive peripheral repertoire and to monitor changes in the frequency of these cells during the development of an autoimmune disease, we generated PLP139–151/IAs tetramers.
In this study, we report generation of PLP139–151/IAs tetramers, which stain PLP139–151-reactive cells with specificity. We have defined optimum conditions for staining using MHC II tetramers specific for a self-Ag. Several factors influence the staining with class II IAs multimers, including the pH of the staining medium and neuraminidase (NA) treatment during the staining reaction. We show that analysis by flow cytometry identifies only a fraction of autoantigen-specific T cells, which are able to interact with specific peptide-MHC complexes as judged by studying the ability of these cells to flux calcium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female SJL mice (4–8 wk of age) were obtained from The Jackson Laboratory (Bar Harbor, ME). They were housed in specific pathogen-free conditions at the animal facility of the Harvard Institutes of Medicine. The mice were kept in accordance with the guidelines set out in the animal protocols of Harvard Medical School. The 5B6 TCR transgenic (Tg) mice specific for myelin PLP139–151 have been described previously (18) and were also housed at the same facility.
Peptides, immunization protocols, and generation of short-term polyclonal T cell lines and 5B6 TCR Tg T cells
The PLP peptides, PLP139–151 (HSLGKWLGHPDKF), PLP178–191 (NTWTTCQSIAFPSK), and Theiler’s murine encephalomyelitis virus (TMEV) VP270–86 (WTTSQEAFSHIRIPLP) were synthesized using F-moc chemistry (QCB BioSource International, Hopkinton, MA). All peptides were HPLC purified. The identity of peptides was confirmed by mass spectroscopy, and they were dissolved in sterile water before use. For immunizations, 100 µg of each peptide was administered emulsified in CFA in multiple sites s.c. Ten days later, the regional lymph nodes were pooled, and single-cell suspensions were obtained after lysing the erythrocytes using 1x ammonium chloride potassium buffer (BioWhittaker, Walkersville, MD). The lymph node cells (LNC) were restimulated in 24-well plates at a density of 5 x 106 cells/ml in complete medium supplemented with 20 µg of the corresponding peptides and APC as described (16). The complete medium consisted of DMEM, 10% FCS, 2 mM L-glutamine, 10 mM HEPES, 2 mM mercaptoethanol, penicillin (1000 U/ml), and streptomycin (1 µg/ml) (BioWhittaker). After 2 days in culture, complete medium containing IL-2 (5 µM; AB Biotechnologies, Columbia, MD) was added into the cultures. These cultures were then restimulated with the peptide (20 µg) and APC every 2 wk, two to three times. The 5B6 Tg T cells specific for PLP139–151 are described elsewhere (18). A T cell line from the LNC of naive 5B6 Tg T cells was generated by repeated stimulation with PLP139–151 as above.
MHC II IAs constructs and generation of soluble MHC molecules and IAs multimeric complexes
The cDNA constructs were made by PCR using plasmids encoding
IAs 
- and 
-chains. The cDNAs encoding the
signal peptides and extracellular domains of the
IAs - and -chains were amplified first, and
the sequence for PLP139–151 peptide
(5'-CATTCTTTGGGAAAATGGCTAGGACATCCCGACAAGTTT-3') was then introduced
in the IAs sequence by overlapping PCR. The
amino acid sequences of the amino-terminal region of the
IAs -chain/PLP139–151
peptide/linker sequences were as follows: MALQIPSLLLSAAVVVLMVLSSPGTEG
(IAs leader); GDSGT (linker); HSLGKWLGHPDKF
(PLP139–151); and GSGSGS (linker). The
IAs carboxyl-terminal residue linked to the
7-aa linker is "K." The sequences corresponding to Fos and Jun
dimerization domains were amplified by PCR from
IAg7 constructs (19). They were
attached to the 3'-end of IAs and
IAs extracellular domains, respectively,
through a 7-aa linker (Val-Asp-(Gly)5) that contained a SalI
restriction site. The amino acid sequences for Fos and Jun linkers were
the following: Fos, VDGGGGG (linker at the amino-terminal end)-Fos
sequence-SAGGG (linker at carboxyl-terminal end); and Jun, VDGGGGG
(linker at the amino-terminal end)-Jun sequence-no linker at the
carboxyl-terminal end. The identities of these constructs were
confirmed by sequencing. The IAs cassette
containing the Fos domain was cloned into the BamHI site of
pAcAB3 vector (BD PharMingen, San Diego, CA), while the
IAs cassette containing the peptide and Jun
dimerization sequence was introduced into the BglII site.
The Bir A site (LGGIFEAMKMELRD) for biotinylation was incorporated into
the 3'-end distal to the Fos sequence in the -chain. To generate
IAs-TMEV70–86, the
sequence for PLP139–151 peptide was excised by
using KpnI and BamHI enzymes and replaced with a
nucleotide sequence encoding TMEV70–86
(5'-TGGACCACCAGCCAGGAAGCGTTCAGCCATATCCGCATCCCGCTGCCG-3').
The soluble MHC molecules were expressed in a baculovirus expression
system by infecting Sf9 insect cells (BD PharMingen). The expression of
MHC - and -chains was confirmed by SDS-PAGE and Western blot
analysis using rabbit polyclonal antisera for Fos and Jun (1:500). To
identify the leucine zippers Fos and Jun, we raised antisera against
them in rabbits using peptides coding for Fos and Jun zippers, which
were kindly provided by Dr. P. Kim (Massachusetts Institutes of
Technology, Cambridge, MA). Large-scale production of soluble MHC
molecules was accomplished by using spinner flasks connected with
spargers (Bellco Biotechnology, Vineland, NJ). The Sf9 cells at a
density of 1 x 106 cells/ml were infected
using a high-titer supernatant of the rIAs
baculovirus (1 x 108 PFU/ml). The cells
were grown in serum-free insect cell medium (BD BaculoGold Max-XP; BD
PharMingen) for 3–5 days. The MHC proteins in the baculovirus-infected
supernatants were concentrated using a Prep/Scale-TFF 1 ft2
cartridge (30k; Millipore, Bedford, MA). The proteins were then
purified on an affinity column prepared using MKS4 mAb (American Type
Culture Collection, Manassas, VA), and the proteins were concentrated
to >5 mg/ml using Centricon-plus 20 columns (Millipore). The
IAs recombinant protein yield was 1
mg/L.
Biotinylation of MHC monomers was conducted at 30°C overnight using biotin protein ligase enzyme at an optimized concentration of 25 µg/10 nmol of substrate according to the manufacturer’s recommendations (Avidity, Denver, CO). The unincorporated biotin was removed by extensive dialysis with 1x PBS (pH 7.5). The multimeric complexes were then generated using streptavidin (SA)-PE (Molecular Probes, Eugene, OR) at different ratios between biotinylated protein and SA-PE ranging from 2:1 to 16:1. Native gels (6% acrylamide) using unconjugated SA confirmed the extent of biotinylation. As a negative control, a tetrameric reagent was also derived for a foreign Ag, TMEV VP270–86 that also binds IAs (20, 21).
T cell proliferative response
Soluble IAs monomers, IAs-PLP139–151 or IAs-TMEV70–86, were coated to 96-well plates overnight in bicarbonate buffer (pH 9.0). The plates were washed twice with 1x PBS, and freshly activated polyclonal T cells specific for PLP139–151, PLP178–191, and TMEV70–86, or 5B6 Tg T cells were added at a density of 2 x 105 cells/well. After 48 h of culture, the cells were pulsed with 1 µCi of [3H]thymidine, and 16 h later, the plates were harvested using a Wallac (Gaithersburg, MD) liquid scintillation counter. The proliferative response was measured as cpm and expressed as mean cpm in triplicate wells.
MHC II IAs tetramer staining based on flow cytometric analysis
Three to 6 days following the activation of polyclonal T cell
lines or 5B6 Tg T cells, the cell suspensions were enriched for T cells
by Ficoll-Hypaque density gradient centrifugation. The cells were
incubated with IAs multimers in DMEM supplemented
with IL-2 (5 µM) at 37°C for 3 h at a concentration of 30
µg/ml. The cells were then washed once with 4 ml of buffer containing
1x PBS, 2% FCS, and sodium azide (0.1%). This was followed by
staining with anti-CD4 Ab (clone RM4.5, CD4-APC) and 7-AAD (BD
PharMingen). After incubating at room temperature (RT) for 20 min, the
cells were washed, as above, and acquired using a FACSort (BD
Biosciences, San Jose, CA). These conditions were used for all of the
tetramer staining reactions, unless otherwise indicated. To define the
optimum conditions for tetramer staining, the following parameters were
studied: concentration of tetramers, duration of staining, temperature
requirement (4–37°C), amount of FCS (0–10%), pH of the staining
medium (DMEM containing IL-2) at the initiation of culture (5.0–9.0),
and the effect of anti-TCRc (clone H57597; BD PharMingen) and
anti-CD3 (clone 145-2C11; BD PharMingen). For all tetramer staining
reactions, we did not purify the multimeric complexes, because there
was no difference between staining with the unpurified or the purified
material obtained from gel filtration columns (Superose 12; Pharmacia,
Piscataway, NJ) (data not shown). The number of tetramer-PE
(PLP139–151 or
TMEV70–86)-positive cells was then determined in
live (7-AAD-negative), CD4-positive populations.
NA treatment
Single-cell suspensions were obtained from the lymph nodes of
SJL mice, and the T cells were enriched by negative selection (mouse
CD3 columns; R&D Systems, Minneapolis, MN). Approximately 1 x
107 cells/ml were used for NA treatment at a
concentration of 0.7 U/ml (NA type X from Clostridium
perfringens; Sigma-Aldrich, St. Louis, MO) in serum-free DMEM at
37°C for 1 h (22). The cells were then washed in
1x PBS and used for flow cytometric analysis. The effect of NA on
lymphocytes was analyzed by forward-scatter (FSC) vs side-scatter (SSC)
profiles, and the expression of surface markers, namely CD3 (clone 145
2C11), CD4 (clone RM4.5), CD25 (clone 7D4), and CD69 (clone H1.2F3) and
their corresponding isotype controls (all FITC-conjugated; BD
PharMingen). The results were then compared between NA-treated and
NA-untreated cell populations. Before tetramer staining, the polyclonal
T cells, which were either rested or freshly activated with peptides
and APC for 5 days, were obtained by Ficoll-Hypaque density gradient
centrifugation and used for NA treatment. The tetramer staining
was conducted on NA-treated cells, as described above, with or without
anti-TCRc (clone H57597) or anti-CD28 (clone 37.51) (BD
PharMingen).
Measurement of calcium fluxing
Flow cytometric analysis.
The T cells enriched from SJL mice immunized with
PLP139–151 were restimulated twice in vitro
using the PLP139–151 peptide and APC. Five days
after the second stimulation, the T blasts were obtained by
Ficoll-Hypaque density gradient centrifugation. The cells at a density
of 106–107 cells/ml were
loaded with the membrane-permeable form (penta-aceto-oxymethyl (AM)
ester) of Indo-1 (2 µg/ml) for 1 h at 37°C in DMEM. Cellular
esterases cleave the AM moiety, resulting in the trapping of Indo-1 in
the cell (23). After washing in 1x PBS, the cells were
resuspended in DMEM to a density of 3 x 106
cells/ml and stored in the dark for 15 min at RT until analysis. The
release of intracellular Ca2+ was determined
using a FACSVantage SE (BD Biosciences). The cytometer was optimized
using ionomycin (2 µg/ml; Sigma-Aldrich) as a positive control such
that the Indo-1 emission ratio of 405 over 490 nm was maximized, and
the ratio was plotted against time. To study the tetramer-induced
response, the cells were acquired for 1 min to obtain the baseline
response, and the IAs tetramers were added into
the cell suspensions in 1 ml at a concentration of 30 µg/ml. The
response was monitored for 9 min. The sample tubes containing the
cells and reagents were kept at 37°C during acquisition of the
data.
Confocal microscopy. The PLP139–151-specific T cell lines were used for measuring the Ca2+ fluxing by confocal microscopy using Fluo-4 which excites at 488 nm and emits at 516 nm. Cells at a density of 1 x 107/ml were loaded with AM esters of Fluo-4 (2 µg/ml), and the mixture was incubated at 37°C for 1 h in DMEM (23). Following washing in 1x PBS, the cells were rested at RT for 15 min in the dark. The adequacy of cell loading was tested by treating a sample with ionomycin (2 µg/ml), and the cell suspension (20 µl) was loaded onto 0.25 mm, express-lane slides (Plus Gold; Grace Bio-Labs, Bend, OR). The fluorescence intensity of the responding cells was measured using a pseudocolor plot in an LSM 510 laser-scanning inverted microscope equipped with argon ion lasers (Zeiss, Heidelberg, Germany). To determine the response for IAs tetramers, the PLP139–151 and TMEV70–86 tetramers were added at a concentration of 30 µg/ml, and the response was measured immediately based on the intensity of pseudocolor (green). The responses to the above stimuli were measured by counting both the number and the intensity of each responding cell by using IPLab software (Sanalytics, Fairfax, VA). The percentage of PLP139–151 tetramer-induced Ca2+ fluxing in the responding cells was calculated as follows: (number of cells responding to PLP139–151 tetramers - number of cells responding to TMEV tetramers)/(number of cells responding to ionomycin - number of cells responding to TMEV tetramers) x 100.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IAs monomers could be successfully
expressed in a baculovirus expression system. Soluble
IAs proteins affinity purified on MKS4 columns
were resolved by SDS-PAGE, and staining with Coomassie blue revealed
two major bands of the expected sizes. These were 40 and 28
kDa, respectively, and represented the IAs
- and -chains. This suggests that the MHC proteins obtained from
the MKS4 Ab column were pure (Fig. 1A). The identity of the bands
was confirmed by Western blotting using the polyclonal anti-Fos and
anti-Jun rabbit antisera, because IAs -
and -chains were linked to Fos and Jun leucine zipper domains,
respectively (Fig. 1B). The presence of
PLP139–151 or TMEV70–86
peptide in the soluble IAs proteins was confirmed
by N-terminal amino acid sequencing (Molecular Biology Core Facility,
Dana-Farber Cancer Institute). The formation of
IAs multimeric complexes was verified on a native
nonreducing gel using unconjugated SA. To form tetramers, the
biotinylated MHC proteins and SA were mixed at ratios ranging from 2:1
to 16:1. The IAs multimers included di-, tri-,
and tetrameric complexes, and only 50–60% of the
IAs monomers was biotinylated (data not shown).
Consistently, the ratios of 4:1 to 8:1 generated the most multimeric
complexes. It has been suggested that the use of the term
"tetramer" is a misnomer, given the fact that higher-order
complexes are present when PE-labeled SA is used for multimerization
(3). Hence, in this article, the terms tetramers and
multimers have been used interchangeably.
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We have provided two lines of evidence to show that the
PLP139–151- or
TMEV70–86-MHC proteins identify T cell
populations specific for appropriate peptide-MHC complexes. First, the
in vitro proliferation assay presented in Fig. 2 confirms that the
IAs monomers stimulate their corresponding
polyclonal T cell lines but not lines specific for another autoantigen.
The specificity of the
PLP139–151-IAs molecule
was further verified by stimulating Tg T cells obtained from a
PLP139–151-specific SB6TCR Tg mouse, and as
expected, activated the 5B6 Tg T cells. These cell lines showed no
response to the control TMEV-IAs tetramers (Fig. 2). As a control, we used polyclonal T cells specific for
PLP178–191 which were not stimulated by either
PLP139–151 or TMEV70–86
monomers confirming that the IAs monomers were
highly specific (Fig. 2). Second, using flow cytometry, the tetrameric
reagents (PLP139–151 or
TMEV70–86) were used to stain various T cell
lines including a polyclonal PLP139–151-specific
T cell line, T cells from the
PLP139–151-specific 5B6 Tg mice, a TMEV
VP270–86-specific T cell line, and a control
polyclonal cell line specific for PLP178–191. As
shown in Fig. 3, TMEV70–86 tetramer stained the TMEV-specific T
cell line and not any other cells, whereas
PLP139–151-IAs tetramer
stained the PLP139–151-specific polyclonal T
cell line and the 5B6 TCR Tg T cells. Neither of the tetramers stained
PLP178–191-specific T cells (Fig. 3). The
PLP139–151 tetramers stained only 46% of 5B6 Tg
T cells. This may be due to a fraction of cells in the Tg T cells that
express endogenous TCR - and/or -chains.
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Because we want to use the tetramers to analyze the evolution of the autoimmune response to PLP139–151 in vivo, we optimized staining conditions to detect maximal numbers of autoreactive cells with specificity. The conditions we examined included the concentration of tetramer, duration of staining, temperature requirement, the presence of FCS, and the pH of the staining reaction (Fig. 4). Optimization was conducted using T cell lines that contain a polyclonal population of Ag-specific CD4 T cells, conditions which will closely mimic those expected in normal SJL mice immunized for the induction of experimental autoimmune encephalomyelitis. As a negative control, the TMEV70–86 tetramer-PE was used and the results were compared between PLP139–151 and TMEV70–86 tetramers.
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Lastly, it has been suggested that coincubation of cells with
anti-TCRc enhances tetramer staining (2). Hence, we
compared the effects of two Ab, namely anti-CD3 (145 2C11) and
anti-TCRc (H57597), on staining with the tetramer. Of these,
while anti-CD3 Ab showed no effect on tetramer staining,
anti-TCRc Ab marginally increased tetramer staining. This was
observed with both PLP139–151-specific
polyclonal and 5B6 Tg T cells (data not shown).
Effect of NA treatment on tetramer staining
NA has been used to enhance staining of CD8 cells by class I tetramers (24). We tested the effect of NA treatment on CD4 cells using the PLP139–151-specific T cell line. We noticed that addition of NA in the reaction mixtures resulted in change in FSC vs SSC profiles, when NA-treated and NA-untreated samples were compared. For example, the percentage of cells in gate 2 in NA-treated samples was increased by 2-fold to 11% as compared with 5% in NA-untreated sample, indicating that there is an increase in both size and granularity of the treated cells. This suggests that the NA treatment may have activated the cell populations (Fig. 5A). Corresponding to this shift, the percentage of cells in gate 1 was reduced in NA-treated sample (74% in NA-treated vs 88% in NA-untreated cultures) (Fig. 5A). To confirm whether the NA treatment activates T cells, the activation status of the cell populations was verified in both NA-treated and untreated populations by staining for the expression of the activation markers (CD25 and CD69). Although the differences in the expression levels of these markers were not statistically significant, the percentage of CD25- and CD69-positive T cells increased in NA-treated cells. Although the percentage of CD4- or CD3-positive T cells did not change significantly between NA-treated and untreated samples, a minor population of CD4high subset was evident in the NA-treated samples (data not shown).
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and
anti-CD28. As shown in Fig. 6, the NA
pretreatment enhanced the tetramer staining for
PLP139–151 by 2-fold in NA-treated cells
(NA-untreated, 2.4%, vs NA-treated, 4.8%). It is also evident that in
all the PLP139–151 tetramer-treated samples, the
CD4 cells appeared to increase in size, presumably due to the
tetramer-induced activation. The addition of anti-TCRc together
with tetramers enhanced the tetramer binding regardless of tetramers
(Fig. 6). Nonetheless, the specificity of the tetramer staining was
still maintained (PLP139–151 tetramer, NA
treatment alone, 3.4%, vs NA-treatment plus anti-TCRc, 6.2%;
TMEV70–86 tetramer, NA-treatment alone, 0.1%,
vs NA-treatment plus anti-TCRc, 0.3%). The effect of
anti-CD28 treatment on tetramer staining was not significant
(Fig. 6).
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The LNC from SJL mice immunized with PLP139–151 were restimulated twice in vitro every 2–3 wk using peptide and APC. In freshly activated T cells, as expected, there was the appearance of two live CD4 populations, namely CD4low and CD4high (Fig. 7). In these subsets, we determined the frequency of PLP139–151-reactive cells in the CD25+ population compared with the frequency of TMEV70–86 tetramer-positive cells. The frequency of PLP139–151 tetramer-positive cells was higher in the CD25+CD4high population (8.6%) compared with that of the CD25+CD4low subset (0.5%). The background staining with TMEV70–86 tetramers was negligible, although also higher in the CD25+CD4high population (0.6%) compared with that of the CD25+CD4low population (0.04%) (Fig. 7). These data are consistent with the expected enrichment of Ag-specific cells in the CD25high subset, as well as with recently reported data showing that activated cells have a CD4high phenotype (3, 25). However, the differences we observed in tetramer staining of rested and activated cell lines led us to investigate whether analyzing cells for a functional interaction with tetramer might be a more sensitive measure of their presence than flow cytometric staining alone.
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We tested whether activating with tetramer could initiate
Ca2+ fluxes in responding T cells and whether
this could be used as a more sensitive measure of the frequency of
detecting autoreactive T cells. This was examined by measuring
intracellular Ca2+ using both flow cytometric
analysis and confocal microscopy. To show that activation with
tetramer-MHC could activate cells specifically, we used Indo-1 for flow
cytometric analysis, which provides a ratiometric measure of
Ca2+ fluxing (23). We observed that
the response occurred in a PLP139–151-specific
cell line in <1 min after the tetramer was added, and this response
persisted for 5 min before returning to resting levels (Fig. 8A). By comparison, the TMEV
tetramer did not induce Ca2+ fluxing over the
basal level in the PLP139–151-specific T cell
line, indicating that the response was specific. This was shown clearly
when the mean Indo-1 ratios of calcium-fluxed responses to
PLP139–151 and TMEV70–86
tetramers were overlaid (Fig. 8A).
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3-fold greater number of
cells than the TMEV-treated sample. These responses were compared with
that of ionomycin (positive control), which is an ionophore, and it
induces Ca2+ response in all the viable loaded
cells. The response to ionomycin was taken as 100%, and this was used
to normalize the responses to the IAs tetramers.
We found that the PLP139–151 tetramer induced
the release of intracellular Ca2+ in 36% of the
total number of cells. In parallel by flow cytometry, only 4% of the
cells were specifically stained by PLP139–151
tetramers. Thus, confocal evaluation allowed us to detect a higher
frequency of Ag-specific T cells than did flow cytometric analysis.
When the mean intensity of the responses was compared between different
stimuli, it was relatively greater with ionomycin than
IAs tetramers (data not shown). However, the mean
intensities of the responses between PLP139–151
and TMEV70–86 tetramers were similar. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and/or -chains of IAs,
with the PLP139–151 peptide to form stable
dimers in the absence of the transmembrane domains. We could
overcome this difficulty by adding the Fos and Jun leucine zipper
domains to the MHC II - and -chains, respectively. Use of these
leucine zipper dimerization domains greatly facilitated assembly of the
heterodimer, an approach that has previously been used for the
expression of soluble IAg7, HLA-DQ8, and HLA-DR2
heterodimers (19, 27, 28).
Several lines of evidence suggest that the tetramers we have generated
are specific for appropriate T cells in that they can activate relevant
cells in a bioassay, stain the cells specifically, and induce
Ca2+ fluxes in the specific T cells. Furthermore,
development of a control tetramer for TMEV will also allow us to
compare self- and foreign Ag-reactive repertoires. As reported by
others (2, 3, 25, 29, 30), the tetramer staining in our
system was best seen in activated cells corresponding to the
CD25CD4high subset. The reason for this
activation dependency for binding of MHC II tetramers to the TCR is not
clear. Conceivably, activation results in reorientation and
configuration of TCR and cell surface molecules leading to an increase
in the avidity of TCR-MHC binding. Whether higher expression of CD4 on
activated T cells further contributes to binding avidity is not clear
yet. But in studies of non-autoantigen-specific tetramers, CD4 has not
been shown to play a critical role in tetramer binding
(1). In agreement with the data published on
anti-TCRc (H57597), we also demonstrated an enhancing effect on
tetramer staining, although in our case the enhancement was marginal
(data not shown). Nonetheless, this effect could still be beneficial,
especially when the rare low-avidity, autoreactive T cells in the naive
repertoire are to be examined. It is not known how anti-TCRc
influences tetramer staining. It is unlikely that the
anti-TCRc-mediated effect can be ascribed to the activation
phenomenon, because the clone 145 2C11, which is also an activating Ab,
when used in parallel with tetramer, did not increase the staining;
rather, it appeared to reduce the tetramer staining in both
PLP139–151 and 5B6 Tg T cell systems (data not
shown).
We evaluated several factors to optimize the conditions for tetramer staining: concentration (30 µg/ml), duration (minimum 3 h), incubation temperature (37°C), reaction medium containing IL-2 without FCS, and pH were all critical in getting maximal staining without increasing the background. Although similar conditions have been described for other MHC II tetramers with respect to concentration, duration, and the temperature requirements (1, 2, 3, 31, 32), we observed in our system that the FCS in the staining medium enhanced nonspecific binding even at low concentrations. To maintain healthy cells in the cultures, we routinely carry out the tetramer staining in DMEM supplemented with IL-2. Similarly, we report here that the pH of the medium appears to be critical for tetramer binding in that the IAs tetramer staining was better when the pH of the medium was relatively alkaline. We tested a wide pH range of the culture medium (pH 5.0–9.0), and the best staining was obtained when the pH was between 8.0 and 8.4 with negligible background staining with the control TMEV IAs tetramer and without compromising cell viability. We opted for pH 8.0 at which the viability of the cells was maximal as determined by 7-AAD staining. We speculate that the alkaline pH may facilitate better accessibility of T cells to the tetramers.
We used NA in our system to test whether it enhances the tetramer staining as demonstrated for the MHC I system (24). We found NA at 0.7 U/ml as optimum based on cell viability analysis using 7-AAD and tetramer staining (data not shown). NA has been used to enhance B cell function, and it acts by removing the sialic acid from the surface of the cells (22, 33). It is suggested that the removal of sialic acid results in a reduction in the net charge of cells in the interacting populations, in our case APC and T cells, which leads to enhanced cell-cell adhesiveness, which is critical for T cell stimulation (22). Our preliminary data suggest that NA treatment may partially activate T cells, and that may be the reason for increased tetramer binding. Our data would suggest that the NA might induce changes in the membrane fluidity and geometry of the surface molecules involved in TCR-MHC-peptide interaction. Although there was no significant increase in CD69 and CD25 expression, the change in size is suggestive of this phenomenon.
In this study, we have demonstrated that the tetramers induce Ca2+ fluxing in an Ag-specific manner which can be detected both by flow cytometry and confocal microscopy. The occurrence of Ca2+ flux following tetramer binding indicates engagement of the TCR by the tetramer, suggesting that the binding of IAs tetramers to the Ag-specific T cells is functional. Given the fact that the binding of MHC II multimers to the autoreactive T cells is poor, the measurement of Ca2+ fluxing may provide a more sensitive measure to identify autoreactive T cells. It is likely that the MHC II tetramers interact with most of the Ag-specific TCRs and induce Ca2+ fluxing even when conventional flow cytometry may not detect these cells. Therefore, it appears from our data that, in a rested polyclonal mixed T cell line, raised against the autoantigen PLP139–151, there is a population which can respond to PLP139–151 tetramer as measured by Ca2+ flux but that is not labeled with the tetramer as detected by conventional flow cytometry. Thus, enumerating and sorting the tetramer-positive cells based on Ca2+ flux may provide an additional tool to the traditional FACS-based staining of Ag-specific cells. The Ca2+ flux together with confocal microscopy will allow phenotypic characterization of responding T cells. In contrast, flow cytometric analysis based on Ca2+ flux will allow one to sort the Ag-specific T cells in mixed cell subsets much more rapidly.
In summary, we have defined an MHC II tetramer system that allows us to detect autoreactive T cells using PLP139–151 as a model self-Ag. In this study, we show that, apart from dose, incubation time, and temperature, the pH of the medium and the presence of FCS are critical for tetramer binding. Though the differences obtained with these changes in the protocol may be marginal, these incremental changes may be especially important when staining the rare Ag-specific T cells in the naive repertoire or in detecting low-avidity autoreactive T cells. We found that the NA treatment enhances the tetramer staining for PLP139–151 substantially, without loss of specificity. Furthermore, we show that the IAs tetramer induces Ca2+ fluxes in an Ag-specific manner and that cells not detected by flow cytometry can flux Ca2+ in response to tetramer binding. Hence, these strategies could be adapted for the detection of rare Ag-specific low-avidity autoreactive T cells in vivo for detecting the evolution of the autoreactive repertoire.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Vijay K. Kuchroo, Room 706, Harvard Institutes of Medicine, Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115. E-mail address: vkuchroo{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: PLP, proteolipid protein; NA, neuraminidase; Tg, transgenic; TMEV, Theiler’s murine encephalomyelitis virus; LNC, lymph node cell; SA, streptavidin; AM, penta-aceto-oxymethyl; RT, room temperature; FSC, forward scatter; SSC, side scatter.
Received for publication September 24, 2002. Accepted for publication November 12, 2002.
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