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CUTTING EDGE |
*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; and
Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In recent reports, MHC tetramer technology has been extended to the class II system (2, 3, 4, 5). These reports used relatively high concentrations of tetramer (20 µg/ml), extended incubation times (1–3 h), and elevated temperatures (22°C or 37°C). A requirement for such conditions would be surprising, given the avidity and fast on-rates expected for the oligomeric MHC-TCR interaction (6, 7). This suggests that class II MHC tetramer staining may reflect a more elaborate process than simple association of a soluble reagent with surface receptors.
In this study, we investigated the utility of oligomers of human class II MHC-peptide complexes in detection of Ag-specific CD4+ T cells. We show by flow cytometry that fluorescent oligomers of DR1 in complex with a peptide from influenza virus specifically stain two DR1-restricted, influenza-specific T cell clones and an Ag-specific polyclonal T cell line, and that the oligomers are internalized efficiently. Treatments that interfere with cytoskeletal rearrangements and endocytosis block class II MHC oligomer staining, showing that an active cellular process is required. Implications of these results for the use of class II MHC oligomers in detection of Ag-specific CD4+ T cells are discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ha306–318 (PKYVKQNTLKLAT), TT830–844 (QYIKANSKFIGITEL), A2103–114 (VGSDWRFLRGYHQYA), and TfR680–696 (RVEYHFLSPYVSPKESP) were synthesized using solid-phase Fmoc chemistry, and purified by C18 reversed phase HPLC. All peptides bind tightly to DR1 with dissociation constants below 100 nM (8).
Preparation of labeled Abs and streptavidin (SA)3
Murine mAbs OKT3 or OKT4 (American Type Culture Collection, Manassas, VA) purified from hybridoma supernatant or SA (ProZyme, San Leandro, CA) were incubated with 10-fold molar excess FITC (Sigma, St. Louis, MO), succinimidyl 6-(biotinamido) hexanoate (NHS-LC-biotin; Pierce; Rockford, IL), or N-(6-(biotinamido)hexyl)-3'-(2'pyridyldithio) propionamide (Biotin-HPDP; Pierce) at pH 7.5 for 3 h at room temperature and isolated by gel filtration using Sephadex G-50 (Pharmacia, Piscataway, NJ). FITC-conjugated SA (SA-FITC) was prepared by preincubation of SA with 2-hydroxyazobenzene-4'-carboxylic acid (Pierce) before fluorescent labeling. R-PE-conjugated SA (SA-PE) was purchased from BioSource International (Camarillo, CA).
Preparation of fluorescent class II MHC oligomers
Soluble HLA-DR1 (B1*0101) peptide complexes carrying an 
subunit C-terminal cysteine (9) were reacted with excess
maleimide-oxyethylene-biotin (PEO-maleimide-activated biotin; Pierce)
or pyridyldithio-propionamide-biotin (HPDP-biotin; Pierce) and isolated
by gel filtration in PBS, pH 7.0. Oligomers were formed by stepwise
addition of SA-FITC or SA-PE (BioSource International). SA-PE from
other sources were tested and found to label cells less brightly.
FITC-labeled tetramers were isolated by gel filtration using sequential
SEC-3000 (Phenomenex, Belmont, CA) and Superdex 200 (Pharmacia)
columns. SA and SA conjugates absorbed to the gel filtration
matrix, but SA-FITC saturated with biotinylated DR1 eluted at its
expected m.w.
T cell clones and line
T cell clones HA1.7 and Cl-1 were cultured as described (9) and rested six or more days before use. A short-term polyclonal CD4+ T cell line was raised by repeated in vitro stimulation of CD8-depleted PBMCs from a DR1-homozygous donor using autologous mitomycin C-treated PBMCs in the presence of 1 µM Ha peptide.
Flow cytometry
T cells (
107/ml) were mixed with
DR1-peptide oligomers for 3–5 h at 37°C, chilled for 5 min,
supplemented with secondary Abs for 30 min, and washed twice with cold
wash buffer (PBS, 1% FBS, 15 mM sodium azide). Inhibitors (stock
solutions in DMSO, ethanol, or PBS; final concentration of solvent
0.5%) were preincubated with cells in PBS for 1 h, after which
the cells were stained with oligomer as above. In cell surface
stripping experiments, chilled and washed cells were resuspended in
wash buffer containing 25 mM 2-mercaptoethanesulfonic acid
(2-ME-SO3-), incubated for 15
min at 37°C, and then washed twice with cold wash buffer. In
inactivation experiments, cells were pretreated overnight in complete
medium with peptide or with immobilized OKT3 (5 µg/ml in PBS, 2
h) and then were stained as above.
Fluorescence microscopy
Live HA.1.7 T cells were isolated using a Ficoll gradient and incubated with 10 mg/ml FITC-dextran (average molecular mass 10 kDa; Sigma) and 70 µg/ml DR1-Ha SA-PE oligomer, or 35 µg/ml SA-PE alone, for 3 h at 37°C, seeded onto cold Cell-Tak (Becton Dickinson Labware, Mountain View, CA)-coated glass coverslips, washed, fixed, mounted in Fluoromount-G (Electron Microscopy Sciences, Fort Washington, PA), and visualized using a DeltaVision digital deconvolution microscope system (Applied Precision, Issaquah, WA).
Dynamic light scattering
Measurements were made at 22°C using a DynaPro-MS/X dynamic light scattering instrument (Protein Solutions, Charlottesville, VA). Protein samples were filtered through 0.2-micron spin filters (Corning-Costar, Cambridge, MA) before analysis. All samples were measured at two different protein concentrations and with identical results. Molecular mass equivalents (in Da) were estimated from hydrodynamic radii (RH, nm) using an empirical model for globular proteins, log MW = 2.426 log (1.549 x RH), as recommended by the manufacturer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The oligomerization strategy used in this work relies on biotin
covalently coupled to a cysteine residue at the C terminus of the
HLA-DR1 subunit (10), with subsequent oligomerization
using SA. Soluble DR1-peptide complexes, folded in vitro from subunits
expressed in Escherichia coli (11), were
biotinylated at the introduced cysteine with >90% efficiency, using a
maleimide reagent carrying biotin at the end of a 29 Å hydrophilic
linker (Fig. 1
A). The
DR1-peptide complexes were resistant to SDS-induced chain dissociation
at room temperature, indicating quantitative peptide loading (Fig. 1A). For routine use, biotinylated DR1-peptide complexes
were oligomerized with SA-PE. Such DR1-SA-PE oligomers carrying the Ha
peptide exhibited Ag-specific binding to two DR1-restricted,
Ha-specific T cell clones, HA1.7 (12) and Cl-1
(13) (Fig. 1, B and C). Oligomers
carrying unrelated peptides showed no significant binding (Fig. 1, B and C, A2 and TfR traces). To confirm the
specificity of oligomer binding we analyzed a series of dilutions of
the HA1.7 clone mixed into nonspecific PBMCs (Fig. 1D).
Monocytes present in the PBMC mixture exhibited nonspecific oligomer
binding, but could be distinguished by their
CD4medium phenotype. The fraction of T cells
staining with the DR1-SA-PE oligomers correlated closely with the
fraction of Ag-specific cells in the mixture, highlighting the
specificity of staining (Fig. 1D).
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We examined the experimental conditions necessary for oligomer
staining of the T cell clones. Both clones required relatively high
oligomer concentration (Fig. 2A), with Cl-1 exhibiting
saturating staining intensity at >300 µg/ml (0.6 µM), whereas
for HA1.7, staining did not appear to saturate even at 750 µg/ml
(2 µM). Staining intensity increased with increasing temperature
for both clones (Fig. 2B). At 4°C, the temperature usually
used for Ab staining, no staining was observed for either clone. The
staining signal developed slowly, and continued to increase for at
least 5 h after the addition of oligomer (Fig. 2C).
These concentration, temperature, and time requirements for efficient
staining with these reagents are consistent with those described by
other researchers using MHC class II oligomers (2, 4, 5).
Our typical staining protocol uses 20–50 µg/ml oligomer reagent for
3–5 h at 37°C.
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D). Treatments that inhibited conventional T cell signaling
pathways, including genistein (14), staurosporine
(15), PP2 (16), and methyl-
-cyclodextrin
(17), weakly inhibited staining or had no effect. Agents
that disrupt endosomal proteolysis, such as chloroquine,
NH4Cl, and monensin, had little or no effect.
However, staining was substantially blocked by treatments that
interfere with endocytosis and cytoskeletal rearrangements, including
the microfilament-disrupting drugs latrunculin A (18) and
cytochalasin D (19), the phosphatase inhibitor
phenylarsine oxide (20), sodium azide (80 mM), and
paraformaldehyde fixation. TCRs are efficiently internalized following
engagement by cell surface MHC-peptide complexes on other cells
(21, 22). Because MHC oligomer staining was blocked by
treatments that block endocytosis, we postulated that oligomers might
be internalized along with TCR during the staining protocol. Bound class II MHC oligomers are present in internalized compartments
To test whether MHC class II oligomers were internalized after
binding, we performed fluorescence microscopy on HA1.7 T cells after
incubation with DR-SA-PE oligomers. Oligomer staining was detected in
intracellular compartments (Fig. 3A, red) colocalized with
endocytic compartments as visualized by FITC-dextran (Fig. 3A, green). Cells incubated with SA-PE reagent alone
(without MHC) showed normal FITC-dextran internalization but no
detectable PE signal (data not shown). These results show that MHC
class II oligomers are internalized efficiently by T cells.
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D), indicating that cell-associated oligomers were not
present at the cell surface. To demonstrate the effectiveness of the
stripping protocol, we examined HA1.7 T cells that had been surface
stained with an Ab carrying the cleavable linker (CD4-SS-bio). In
this experiment, staining was limited to the cell surface by incubation
at 4°C. Fluorescent SA was efficiently stripped from the SS-bio Ab by
the 2-ME-SO3- treatment (Fig. 3B) but not from a noncleavable biotinylated Ab (Fig. 3C). The insensitivity of the oligomer-binding signal to the
2-ME-SO3- surface stripping
protocol and the lack of surface staining observed by fluorescence
microscopy both indicate that essentially all of the oligomers
associated with T cell staining are present in internal
compartments.
One possibility for the correlation between MHC oligomer staining and
internalization might be a low valency in preparations of DR1-SA-PE.
The actual oligomeric state of MHC-SA-PE oligomers has not been
reported. Moreover, physical characterization is difficult due to the
large size of the PE moiety (250 kDa) and heterogeneous
cross-linking in commercial preparations of SA-PE. To obtain MHC
oligomers with a well-defined valency, we prepared oligomers using SA
labeled with FITC. Because several preparations of commercially
available SA-FITC conjugates exhibited substoichiometric biotin-binding
capacity, we developed a strategy to prevent damage to the
biotin-binding sites during FITC labeling by protecting the
biotin-binding sites using the weakly binding biotin analog
2-hydroxyazobenzene-4'-carboxylic acid (see Materials and
Methods). DR1-SA-FITC oligomers prepared with this reagent
exhibited an average molecular mass (230 kDa) consistent with one SA
(60 kDa) and four DR1 (45 kDa), indicating that the desired tetrameric
species had been formed (Fig. 3G). Gel filtration analysis
gave a similar result (265 kDa, data not shown). By contrast, DR1-SA-PE
oligomers exhibited a heterogeneous population distributed around a
molecular mass of 15,000 kDa (Fig. 3F), indicating that they
are composed of multiple SA and/or PE moieties; they are either large
oligomers or noncovalent aggregates and, properly, they should not be
referred to as "tetramers." T cell staining by the defined
DR1-SA-FITC tetramers (Fig. 3E) was considerably less
intense than that observed for the DR1-SA-PE oligomers (Fig. 3D), even after expression of the staining results in terms
of numbers of fluorophores bound per cell (data not shown).
Nonetheless, the observed staining still required elevated temperature
and was insensitive to surface stripping by
2-ME-SO3- (Fig. 3E).
Thus, the observed association of staining and endocytosis was not due
to a reduced valency in the DR1-SA-PE oligomers.
A polyclonal T cell line contains subpopulations with different requirements for staining
To test the generality of the observation that staining with MHC
class II oligomers requires an active cellular process, we tested a
polyclonal T cell line restricted by HLA-DR1 and specific for the Ha
peptide. Oligomer staining experiments were performed in parallel at
37°C and at 4°C (Fig. 4A).
At 37°C, 67% of the total polyclonal cell population exhibited
DR1-SA-PE staining (PE+), which was specific for
the appropriate peptide. By contrast, only 12% of the cells were
PE+ when stained at 4°C, and these exhibited
2-fold reduced intensity. Both the minor PE+
population detected at 4°C and the major population detected at
37°C were oligoclonal, as shown by TCR V3 and C1 analysis (data
not shown), and both had characteristics of CD4+
memory T cells (CD3+, CD4+,
CD8-, CD25+,
CD45RO+, and CD62L-).
These results show that most of the polyclonal T cells share with HA.17
and Cl-1 the requirement for an active cellular process to observe MHC
oligomer staining, although some cells can stain in the absence of such
processes.
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T cells can enter a nonresponsive or anergic state in response to
a partial activation stimulus (23). We tested whether such
treatments would effect T cell staining by MHC oligomers. Treatment
with high concentrations of antigenic but not control peptides, or with
immobilized anti-CD3, each dramatically reduced oligomer staining
of the polyclonal T cell line (Fig. 4B). These treatments
are known to induce T cell energy (24, 25). TCR surface expression was
reduced somewhat by these treatments (Fig. 4C), but the
effect was much smaller and accounted for <10% of the overall
reduction in oligomer staining (Fig. 4B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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10-6 M
(10) within the range observed for other MHC-TCR
interactions (10-4–10-7)
(2, 6). In addition, a temperature dependence of staining
similar to that observed here has been reported previously for murine
class II MHC oligomers (2). Finally, similar behavior was
observed in a short-term T cell line as well as in two Ag-specific T
cell clones. Thus, a requirement for active cellular processes
resulting in oligomer internalization may be a general (although not
universal) characteristic of oligomer staining in the class II MHC
system. The requirement for active processes and association with internalization can be understood in terms of the effects of multivalent engagement of TCR. The MHC-TCR interaction is relatively weak, and would be expected to require multivalent engagement to survive the washing steps required for flow cytometry. Indeed, such considerations led to the initial development of MHC tetramers as staining reagents (26). Multivalent engagement is likely to require reorganization or rearrangement of TCR molecules in the plane of the membrane (10). Such rearrangements are likely to require cytoskeletal participation (27) and could be altered in nonresponsive or anergic T cells (28). For CD4+ T cells, multivalent engagement will trigger activation processes (9, 29, 30) leading to down-regulation (internalization) of activated TCR (22, 30) and internalization of bound MHC oligomers.
Staining of CD8+ T cells by class I MHC tetramers in general does not appear to require receptor internalization or other active T cell processes, as evidenced by their ability to be stained at 4°C (26, 31, 32) (although internalization has been reported to increase staining intensity and specificity; Ref. 33). The reason for this difference between the behavior of class I and class II MHC oligomers is not clear. It may reflect differing roles for the coreceptors CD8 and CD4 in oligomer binding or cellular activation, or an intrinsic difference in the ability to cross-link or aggregate TCRs in CD8+ vs CD4+ T cells.
One of the primary attractions of using MHC tetramers to detect Ag-specific T cells has been their ability to bind T cells independent of cellular response or activation state. This has been observed for class I MHC tetramers in several studies (26, 34, 35). If the activation dependence of class II MHC oligomer staining is a common feature of the system, it may preclude the use of class II MHC oligomers to characterize naive, inactive, or anergized CD4+ T cells. These considerations may be relevant to recent reports using class II MHC oligomers to investigate responding frequencies for CD4+ T cells (3, 4, 5).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lawrence J. Stern, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge MA 02139.
3 Abbreviations used in this paper: SA, streptavidin; SA-FITC, FITC-conjugated SA; SA-PE, R-PE-conjugated SA; 2-ME-SO3-, 2-mercaptoethanesulfonic acid.
Received for publication August 17, 2000. Accepted for publication November 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) cells. Standard conditions (50 µg/ml, 37°C, 5 h) were
varied as noted. D, Inhibitor analysis. Fraction of
signal observed in the treated relative to an untreated sample is shown
(+, <40%; +/-, 40–70%; -, >90%).
, oligomer staining intensity; 
,
staining after normalization for CD3 expression. C, MHC
oligomer and CD3 staining levels after pretreatment with 10 µM
control (TfR) or specific (Ha) peptide.