Functional Analysis of B and T Lymphocyte
Attenuator Engagement on CD4 + and CD8+
T Cells
This information is current as
of June 17, 2017.
Carsten Krieg, Peggy Han, Roslynn Stone, Olivia D.
Goularte and Jonathan Kaye
J Immunol 2005; 175:6420-6427; ;
doi: 10.4049/jimmunol.175.10.6420
http://www.jimmunol.org/content/175/10/6420
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References
The Journal of Immunology
Functional Analysis of B and T Lymphocyte Attenuator
Engagement on CD4ⴙ and CD8ⴙ T Cells1
Carsten Krieg, Peggy Han, Roslynn Stone, Olivia D. Goularte, and Jonathan Kaye2
T
cell specificity results from TCR-mediated recognition of
specific Ag in conjunction with MHC on APCs (1). However, the functional outcome of this initial TCR engagement is modulated by secondary signals, which can have costimulatory or coinhibitory functions. The group of coinhibitory
molecules includes CTLA-4 and programmed cell death-1 interacting with CD80 and CD86 or PD-L1 and PD-L2, respectively
(reviewed in Ref. 2).
We recently cloned a gene from differentiating thymocytes that
encodes a member of the Ig superfamily of cell surface proteins
(3). This protein, independently isolated from Th1 cells, has been
named B and T lymphocyte attenuator (BTLA)3 (4). BTLA is most
highly expressed by B cells, followed by T cells and APC (3),
although its expression on macrophages has recently been reported
to differ between mouse strains (5). The cytoplasmic tail of BTLA
contains ITIM motifs, suggesting an inhibitory function for the
protein. Indeed, lymphocytes from BTLA-deficient mice were
found to be hyper-responsive to anti-CD3-mediated proliferation
(3, 4). In contrast to CTLA-4 and programmed cell death-1, which
are induced during T cell activation, BTLA is constitutively expressed on naive T cells and is further up-regulated upon T cell
activation (3). Although BTLA was thought originally to be an
additional member of the B7 family-recognizing T cell surface
proteins, it has recently been demonstrated that herpes virus entry
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
mediator (HVEM), a known ligand for the TNF family member
LIGHT (derived from homologous to lymphotoxin, inducible expression), is the ligand for BTLA (6, 7). Like BTLA, HVEM is
expressed by T and B cells although the hierarchy of expression on
naive cells is reversed from that of BTLA (i.e., T⬎B) (8). A soluble form of HVEM has been shown to inhibit anti-CD3-induced
proliferation of CD4⫹ T cells, and this effect could be reversed by
anti-BTLA Ab (7). In addition, T cells from HVEM-deficient mice
are hyperproliferative in response to Con A (9). Together, these
data are consistent with a coinhibitory signal mediated by a BTLAHVEM interaction.
We previously generated a panel of BTLA-specific mAb that
preferentially react with the BTLA.2 allelic form of the protein (3).
In this study we further characterize the functional properties of
one of these Abs, PK18. This Ab has agonistic properties that
allow analysis of the effects of BTLA activation and proliferation
of naive CD4⫹ and CD8⫹ T cells.
Materials and Methods
Animals
C57BL/6J (B6), BALB.K, BTLA-deficient (BTLA⫺/⫺), and AND TCRtransgenic (Tg) mice on an H-2b background (10) were used in these studies. All animals were bred at The Scripps Research Institute and maintained
under specific pathogen-free conditions. Experiments were conducted in
accordance with National Institutes of Health Guidelines for the Care and
Use of Animals and with an approved animal protocol from The Scripps
Research Institute animal care and use committee.
Received for publication April 28, 2005. Accepted for publication August 19, 2005.
Abs and flow cytometry
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.
BTLA-specific rat mAb PK3 (IgM␬), PK18 (IgG1␬), and mouse mAb
PJ196 (IgG1␬) were used in these studies. The production of mAb was
described previously (3). The following additional Abs were used (all from
eBioscience, unless stated otherwise): FITC- or APC-conjugated anti-CD8
(clone 53-6.7), PE-conjugated anti-CD3 (clone 145-2C11), PE- or allophycocyanin-conjugated anti-CD4 (clone RM4-5), biotinylated anti-CD69
(clone 1.2F3), and allophycocyanin-conjugated anti-CD25 (clone PC61.5).
Secondary staining reagents included PE-conjugated anti-rat IgM (clone
G5-238; BD Pharmingen) and PE-labeled streptavidin (Biomeda). Cell surface staining was performed as described previously (3). In some instances
cells were preincubated with anti-CD16/CD32 to block FcRs. For the study
of T cell apoptosis, the annexin V-FITC apoptosis detection kit was used
1
This work was supported by National Institutes of Health Grant AI31231. This is
Manuscript 17365-IMM from The Scripps Research Institute.
2
Address correspondence and reprint requests to Dr. Jonathan Kaye, Department of
Immunology IMM-8, The Scripps Research Institute, 10550 North Torrey Pines
Road, La Jolla, CA 92037. E-mail address: [email protected]
3
Abbreviations used in this paper: BTLA, B and T lymphocyte attenuator; HVEM,
herpes virus entry mediator; LIGHT, derived from homologous to lymphotoxin, inducible expression; Tg, transgenic; Treg, regulatory T cell; YFP, yellow fluorescent
protein; wt, wild type.
Copyright © 2005 by The American Association of Immunologists, Inc.
0022-1767/05/$02.00
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T cell activation can be profoundly altered by coinhibitory and costimulatory molecules. B and T lymphocyte attenuator (BTLA)
is a recently identified inhibitory Ig superfamily cell surface protein found on lymphocytes and APC. In this study we analyze the
effects of an agonistic anti-BTLA mAb, PK18, on TCR-mediated T cell activation. Unlike many other allele-specific anti-BTLA
mAb we have generated, PK18 inhibits anti-CD3-mediated CD4ⴙ T cell proliferation. This inhibition is not dependent on regulatory T cells, nor does the Ab induce apoptosis. Inhibition of T cell proliferation correlates with a profound reduction in IL-2
secretion, although this is not the sole cause of the block of cell proliferation. In contrast, PK18 has no effect on induction of the
early activation marker CD69. PK18 also significantly inhibits, but does not ablate, IL-2 secretion in the presence of costimulation
as well as reduces T cell proliferation under limiting conditions of activation in the presence of costimulation. Similarly, PK18
inhibits Ag-specific T cell responses in culture. Interestingly, PK18 is capable of delivering an inhibitory signal as late as 16 h after
the initiation of T cell activation. CD8ⴙ T cells are significantly less sensitive to the inhibitory effects of PK18. Overall, BTLA adds
to the growing list of cell surface proteins that are potential targets to down-modulate T cell function. The Journal of Immunology, 2005, 175: 6420 – 6427.
The Journal of Immunology
according to manufacturer’s instructions (BD Pharmingen). Briefly, T cells
were washed with ice-cold PBS (Invitrogen Life Technologies), resuspended in annexin V-binding buffer (10 mM HEPES/NaOH (pH 7.4), 140
mM NaCl, and 2.5 mM CaCl2), stained with annexin V-FITC and -propidium iodide, and assayed within 1 h by flow cytometry. As a positive
control, T cell apoptosis was induced by dexamethasone (100 nM; SigmaAldrich). At least 20,000 viable cells were live-gated on a FACSCalibur or
digital LSRII using CellQuest software (BD Biosciences) and were analyzed using FlowJo software (TriStar).
T cell preparation
CFSE labeling
Total T cells were washed with PBS, resuspended at a density of 1 ⫻ 107
cells/ml in PBS containing 2 ␮M CFSE (Molecular Probes), and incubated
for 5 min at room temperature. Labeling was terminated by addition of FCS to
a final concentration of 10%, and cells were washed once in culture medium.
The frequency of CD4⫹ or CD8⫹ T cell precursors that were activated to
divide was calculated as the number of CFSE⫹ cells in each division/2n,
where n is the number of divisions, divided by the input number of cells.
T cell activation
A total of 1 ⫻ 106 T cells/ml were cultured in 100 ␮l or 1 ml in 96- or
24-well, flat-bottom plates, respectively, coated with 1:1 ratios of anti-CD3
(clone 145-2C11; eBioscience) in combination with rat IgG (Jackson ImmunoResearch Laboratories), PK18, or PJ196 at the indicated total Ig concentration. In some experiments, T cells were stimulated with a combination of 50 ng/ml PMA and 500 ng/ml ionomycin (Sigma-Aldrich). For
analysis of Ag-specific T cell proliferation, CD4⫹ T cells were cultured at
5 ⫻ 105 cells/ml with 2 ⫻ 106 cells/ml irradiated (3300 rad) BALB.K
splenocytes in the presence of 100 nM pigeon cytochrome c peptide. For
proliferation studies, T cells were cultured in 96-well plates for a total of
72 h and pulsed with 1 ␮Ci/well [3H]thymidine (PerkinElmer) for the last
16 h. Cells were harvested onto glass-fiber filters (Brandel), and [3H]thymidine incorporation was determined. To analyze T cell activation markers, cultures were harvested at various times and were analyzed by flow
cytometry. In other experiments, CFSE-labeled total T cells were stimulated for 72 h and then analyzed.
IL-2 ELISA
Culture supernatants were assayed for mouse IL-2 using a standard sandwich ELISA (eBioscience). Maxisorb plates (Nunc) were coated with antiIL-2 mAb (clone JES6-1A12), and detection was performed using the corresponding biotinylated mAb. The plates were developed using
streptavidin-HRP and tetramethxylbenzidine as substrate. Plates were read
on a plate reader at 450 nm, and data were analyzed using Softmax software (reader and software from Molecular Devices). Samples were performed in duplicate. The assay was standardized with recombinant murine
IL-2, and detection ranged from 1 to 1000 pg/ml.
Cell transfection and mutagenesis
Jurkat T cells were transfected with a construct encoding murine BTLAyellow fluorescent protein (YFP) fusion protein (base vector pEYFP-N1;
BD Clontech) by electroporation and were stained and analyzed by flow
cytometry on day 2. PK18 does not stain untransfected Jurkat T cells.
Site-specific mutations were made using the GeneTailor Site-Directed Mutagenesis System (Invitrogen Life Technologies) according to the manufacturer’s instructions. All mutant constructs were sequence verified (TSRI
Center for Nucleic Acids Research).
Results
Characterization of BTLA-specific mAb
We previously generated BTLA-specific mAb by immunizing rats
or mice with a recombinant soluble form of the protein (3). All Abs
generated reacted preferentially with BTLA expressed on cells derived from B6, but not BALB/c, mice, the results of allelic variation in the BTLA external domain (3). We also demonstrated that
coimmobilized rat anti-BTLA mAb PK18 is a potent inhibitor of
anti-CD3 Ab-induced T cell proliferation (3) (Fig. 1A). Soluble
anti-BTLA Ab PK18 also significantly inhibited proliferation of
CD4⫹ T cells at limiting concentrations of plate bound anti-CD3
Ab (Fig. 1B). As a control, PK18 had no effect on the proliferation
of CD4⫹ T cells derived from BTLA-deficient mice (Fig. 1B). The
PK18-mediated inhibition of T cell proliferation is not due to altered kinetics of the response (Fig. 1C). Seven additional mAb
(one rat- and six mouse-derived) had no effect on (or, in some
experiments, enhanced) T cell proliferation (for example, Ab
PJ196 in Fig. 1A). The inability of other anti-BTLA Abs to inhibit T
cell proliferation is not due to poor binding. In fact, PJ196 stains cells
brightly even at extremely low concentrations of Ab, whereas PK18
is, somewhat surprisingly, a relatively poor staining reagent (data not
shown). This suggested that PK18 and other Abs, such as PJ196,
might differ in the respective epitopes on BTLA recognized.
We took advantage of the ability of PK18 and the other mAb to
distinguish BTLA.2 (B6-derived) and BTLA.1 (BALB/c-derived) to
map residues in the external domain of BTLA that are important for
binding. BTLA.2 and BTLA.1 differ in 11 aa in the external domain
of the cell surface protein (positions 41, 45, 47, 52, 55, 63, 74, 85, 91,
102, and 143, with the start methionine designated position 1). Murphy et al. (5) reported that residue 74 was an asparagine in both B6and BALB/c-derived BTLA, whereas other strains had a histidine at
this position. Our BALB/c-derived clone also had a histidine at this
position, but whether this reflects differences in substrains is not
known. A construct encoding a fusion of BTLA.2 and YFP was subjected to site-directed mutagenesis to allow the expression of mutants
of BTLA.2-YFP that had individual residues changed to those in
BTLA.1 (with the exception of codons encoding residues 45 and
47, which were mutagenized together). Individual BTLA-YFP
constructs were transfected into Jurkat cells, the cells from each
transfection were aliquoted for staining with various BTLA mAb,
and the cells were subjected to two-color FACS analysis.
The cell surface expression of BTLA-YFP was quite variable
between individual transfected cells despite similar expression of
YFP (data not shown). Whether this reflects tight regulation of cell
surface transport of the BTLA protein or a property of this particular fusion protein is not known. Nevertheless, cells were gated for
a narrow range of expression of YFP, and then anti-BTLA mAb
staining was assessed. All 10 mutant proteins were expressed on
the cell surface (i.e., at least one anti-BTLA Ab could bind at near
wild-type (wt) levels), but only three had significantly reduced
PK18 staining compared with cells expressing wt BTLA-YFP
(Fig. 1D and data not shown). Mutation of residue 41 from
BTLA.2 to BTLA.1 (P41E) completely abolished binding of
PK18, whereas C85W and T45N/T47K mutations had lesser effects on Ab binding. Binding of PJ196 to mutant 45/47 was also
reduced, as was binding to mutant 41, although to a somewhat
lesser extent. A more modest reduction in binding of PJ196 to the
85 mutant was also observed. Staining of Ab PK3 to all three
mutants is also shown for comparison, with little reduction in
staining to mutants 41 and 45/47, and modest reduction in staining
to mutant 85 (Fig. 1D). Together, the data indicate that PK18 and
PJ196 have overlapping, but distinct, epitopes that probably account for their distinct biological activities.
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All culture experiments were performed in Click’s medium (Irvine) containing 10% heat-inactivated FCS (Invitrogen Life Technologies), penicillin/streptomycin (Invitrogen Life Technologies), 200 mM glutamine, and
50 ␮M 2-ME (Sigma-Aldrich). In some cultures anti-CD28 (clone 37.51;
eBioscience) was used at a concentration of 10 ␮g/ml, or recombinant
murine IL-2 (eBioscience) was added at various concentrations. Cells were
cultured under standard conditions at 37°C in 5% CO2. T cells from spleens
or lymph nodes were enriched by negative selection using anti-B220 (clone
RA3-6B2; eBioscience), anti-Ab class II MHC (clone Y3P) Abs, and antirat IgG magnetic beads (Qiagen). For Ag-specific proliferation assays,
AND TCR-Tg mice were used as an enriched source of naive CD4⫹ T cells.
For CD4⫹ T cell enrichments anti-CD8 (clone 53-6.7; eBioscience), Ab was
added to the mixture. The purity of the resultant cell populations was ⬎95%.
In some experiments, naive CD4⫹CD25⫺ T cells were purified by sorting on
a FACSDiva or FACSAria (BD Biosciences) to a purity of ⬎99%.
6421
6422
FUNCTIONAL ANALYSIS OF BTLA ENGAGEMENT ON T CELLS
Differential effects of BTLA-specific mAb on CD4⫹ and CD8⫹ T
cell proliferation
To better understand the functional consequences of BTLA engagement on CD4⫹ and CD8⫹ T cells, we took advantage of the agonistic
ability of the PK18 mAb. T cells were labeled with CFSE and stimulated for 3 days with anti-CD3 in combination with PK18 or control
rat IgG. The CFSE profile of total T cells revealed an almost complete
block of T cell proliferation, consistent with results obtained by thymidine uptake (Fig. 2A). However, when CD4⫹ and CD8⫹ T cells
were individually analyzed in these assays, there was a near complete
block of CD4⫹ T cell proliferation, whereas a significant proportion of CD8⫹ T cells had divided (Fig. 2A). From these data we
also calculated the precursor frequency of cells that were activated
to divide in these cultures (Fig. 2A). Only 2.2% of CD4⫹ precursor
T cells underwent proliferation when activated by anti-CD3 in the
presence of PK18, compared with 33.3% in control cultures. More-
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FIGURE 1. Characterization of antiBTLA mAb. A, T cells were cultured in
plates coated with equal concentrations
of anti-CD3 and rat IgG (f), anti-CD3
and PK18 (䡺), or anti-CD3 and PJ196
(Œ). Concentrations refer to total IgG.
Data are expressed as the mean
[3H]thymidine incorporation of triplicate cultures (⫾SD) minus the cpm of
T cells cultured alone. B, CD4⫹ T cells
isolated from BTLA⫺/⫺ mice (top
graph) or B6 mice (bottom graph)
were stimulated with the indicated concentrations of plate-bound anti-CD3 in
the presence of soluble rat IgG (f) or
soluble PK18 (F) at a concentration of
60 ␮g/ml. Proliferation was determined
by [3H]thymidine incorporation, as described in A. C, T cells were stimulated
with anti-CD3 and rat IgG (f) or antiCD3 and PK18 (䡺). [3H]Thymidine
incorporation was determined at the indicated times as described in A. D,
Jurkat T cells were transfected with
constructs encoding a fusion of the wt
or mutant BTLA.2 extracellular domain and YFP. Cells were stained with
PK3, PK18, or PJ196 mAb as indicated
and were analyzed by flow cytometry.
The histograms shown are gated on a
narrow range of YFP expression. Isotype control staining is shown as thin
lines. Results are representative of
three experiments.
over, the few CD4⫹ cells that did proliferate underwent a single
division after anti-CD3 and PK18 stimulation, compared with up
to three divisions in control cultures. In contrast, 57.9 and 28.5%
of CD8⫹ precursors proliferated in the presence of anti-CD3 and
rat IgG or PK18, respectively, and some cells underwent three or
four divisions even in the presence of PK18.
Both CD4⫹ and CD8⫹ T cells express BTLA, although CD4⫹ T
cells have modestly higher cell surface expression (3). In addition,
TCR-mediated activation of T cells up-regulates BTLA (3). Thus, the
more profound inhibition of proliferation of CD4⫹ compared with
CD8⫹ T cells could result from differences in expression. To address
this, we stained T cells activated by anti-CD3 in the presence or the
absence of anti-CD28-mediated costimulation for expression of
BTLA (Fig. 2B). Up-regulation of BTLA was detected as early as day
1 after activation of CD4⫹ T cells and reached maximal levels by day
3. Costimulation had little effect early, but overall produced an
The Journal of Immunology
6423
increase in the number of responding cells. A similar pattern was
observed for CD8⫹ T cells, although up-regulation lagged behind that
in CD4⫹ T cells, and the level of expression was ⬃2-fold lower.
Thus, differential effects of PK18 on the proliferation of CD4⫹ or
CD8⫹ T cells could, at least in part, result from differential amounts
of cell surface protein and differing kinetics of up-regulation of BTLA
surface expression. However, the fact that anti-BTLA can inhibit cytokine production before BTLA up-regulation (see below) suggests
that this may not be the only factor involved in the differential effects
of BTLA engagement on CD4⫹ and CD8⫹ T cells.
It was possible that the failure of CD4⫹ T cells to proliferate in the
presence of PK18 was due to the induction of cell death. However,
no significant loss of cell number was observed in PK18-treated
cultures (data not shown). In addition, no increase in apoptotic
cells as assessed by propidium iodide/annexin V staining was observed after activation of CD4⫹ T cells for 6 h with anti-CD3 in
the presence of PK18 compared with that in cells activated in
control cultures (Fig. 3A). Analysis 16 h after stimulation yielded
similar results (data not shown).
We have previously reported that BTLA is expressed by
CD4⫹CD25⫹ T cells, a subset that includes regulatory cells (Tregs)
(reviewed in Ref. 11). Thus, we also considered the possibility that
PK18 was activating Tregs, which then acted to suppress the proliferation of CD4⫹ T cells. To test this, we purified CD4⫹CD25⫺
T cells and tested the effect of BTLA engagement on anti-CD3mediated proliferation as before. As observed for unseparated
CD4⫹ T cells, proliferation of CD4⫹CD25⫺ T cells was inhibited
by PK18 (Fig. 3B). Thus, the ability of anti-BTLA to inhibit CD4⫹
T cell proliferation is a direct effect on the CD4⫹ T cell itself.
other T cell activation markers. Interestingly, the induction of CD69
expression on both CD4⫹ and CD8⫹ T cells was not inhibited by
PK18 (Fig. 4). In contrast, BTLA engagement led to inhibition of
subsequent IL-2R (CD25) expression on both CD4⫹ and CD8⫹ T
cells (Fig. 4). However, the decrease in CD25 expression was more
pronounced on CD4⫹ T cells, consistent with the greater sensitivity of
this population to PK18-mediated inhibition of proliferation. The increased frequency of CD25⫺ T cells, particularly pronounced among
CD8⫹ T cells, was due to reduced proliferation in the presence of
PK18 and, thus, increased frequency of unactivated cells in these cultures. Because IL-2 is necessary for T cell proliferation in vitro as well
as for maintenance of IL-2R expression (12), these results suggested
that the primary defect in anti-BTLA engagement could be cytokine
production. Indeed, anti-CD3- and PK18-stimulated T cells showed a
80-fold reduction in IL-2 secretion compared with anti-CD3- and rat
IgG-stimulated T cells (Fig. 5A).
To determine whether the decrease in IL-2 production was the sole
cause of the inhibition of T cell proliferation in these cultures, we
performed add-back experiments with exogenous IL-2 (Fig. 5B). The
addition of 80 pg/ml recombinant murine IL-2, comparable to that
produced by anti-CD3-activated T cells themselves in these cultures
(Fig. 5A), was not sufficient to rescue the proliferation of T cells in the
presence of PK18 (Fig. 5B), nor did this concentration of IL-2 restore
CD25 to wt levels (Fig. 5C). Thus, the failure to produce IL-2 is not
the sole cause of T cell inhibition mediated by PK18 under these
conditions. Nevertheless, high concentrations (800 pg/ml) of IL-2 are
sufficient to restore nearly 70% of the T cell proliferative response
achieved in control cultures (Fig. 5B). Despite this, high concentrations of IL-2 were not sufficient to return the expression of CD25 to
wt levels on CD4⫹ or CD8⫹ T cells activated in the presence of
PK18, although, again, the effects of PK18 were more pronounced on
the CD4⫹ T cell population (Fig. 5C).
IL-2 production, but not CD69 induction, is suppressed by
BTLA engagement
CD28 costimulation partially overcomes inhibition of T cell
activation and proliferation by BTLA engagement
Induction of proliferation is a relatively late event after T cell activation. To determine whether PK18 blocked all signals mediated by
TCR engagement, we asked whether PK18 prevented the induction of
CD28 costimulation is a strong enhancer of IL-2 production in T
cells (13) and thus might be expected to overcome PK18 inhibition. At low concentrations of anti-CD3, PK18 was still inhibitory,
PK18-mediated inhibition of T cell activation does not involve
regulatory T cells or induction of apoptosis
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FIGURE 2. Differential effects of BTLA engagement on CD4⫹ and CD8⫹ T cells. A, CFSE-labeled T cells were cultured in the presence of anti-CD3
and rat IgG (u) or anti-CD3 and PK18 (f). Plate-bound Abs were used at 20 ␮g/ml total Ig. On day 3 of culture, cells were stained for CD4 or CD8 and
were assayed by flow cytometry. The distributions of precursor cell divisions in anti-CD3 and rat IgG (u) or anti-CD3 and PK18 (f) were also calculated.
One representative experiment of four is displayed. B, T cells were cultured in medium (u) or were stimulated for various times, as shown, with anti-CD3
(bold lines) or anti-CD3 and anti-CD28 (dotted lines). Thin lines and vertical bars indicate isotype controls. T cells were stained for CD4, CD8, and BTLA,
the latter using the PK3 mAb, and were analyzed by flow cytometry. One representative experiment of three is shown.
6424
FUNCTIONAL ANALYSIS OF BTLA ENGAGEMENT ON T CELLS
even in the presence of CD28 (Fig. 6A). However, at higher concentrations of anti-CD3, addition of CD28 costimulation restored
T cell proliferation to near normal levels even in the presence of
PK18 (Fig. 6A). Similar results were found when tracking individual divisions of CFSE-labeled CD4⫹ and CD8⫹ T cells stimulated with saturating amounts of anti-CD3 (Fig. 6B). Under these
conditions, CD28 costimulation was also able to restore CD25
expression on CD4⫹ and CD8⫹ T cells activated in the presence
of PK18 (Fig. 6C). As expected, the addition of anti-CD28 increased IL-2 production in these cultures ⬃50-fold (Figs. 5A and
6D). Thus, the ability of anti-CD28 to overcome PK18 inhibition
mirrors that observed after adding high concentrations of exogenous IL-2 (Fig. 5C). However, even in the presence of costimulation and a high concentration of anti-CD3, the presence of PK18
caused a nearly 13-fold reduction in IL-2 secretion (Fig. 6D).
Thus, CD28-mediated costimulation does not ablate the inhibitory
signal initiated by BTLA engagement.
Given that soluble PK18 could inhibit the proliferation of CD4⫹
T cells at limiting concentrations of plate-bound anti-CD3 in the
presence of costimulation, we also tested the effect of PK18 on
Ag-stimulated CD4⫹ T cells. AND TCR-Tg CD4⫹ T cells were
stimulated in the presence of irradiated BALB.K splenocytes as a
source of APCs. We have previously shown that BTLA is expressed on T cells and APC, and that PK18 binds preferentially to
B6-derived splenocytes, but not BALB.K splenocytes (3). Thus, in
these experiments PK18 would target the T cells and not the APC.
Addition of PK18 to these cultures significantly inhibited T cell
proliferation in response to peptide Ag (Fig. 6E).
BTLA signal can inhibit proliferation of preactivated T cells
Our results demonstrated that BTLA engagement at the time of
initial TCR activation was a potent inhibitor of aspects of T cell
activation. However, the fact that CD69 induction was not affected
by PK18 raised the possibility that the inhibitory effect of BTLA
engagement might be a relatively late event. Thus, we isolated
naive CD4⫹ T cells from B6 mice, stimulated them overnight with
FIGURE 4. PK18 inhibits the expression of CD25, but not CD69. T
cells were stimulated, as described in Fig. 2, with anti-CD3 and rat IgG (u)
or anti-CD3 and PK18 (bold lines). Shown is expression of CD69 or CD25
after 16 or 48 h in culture, respectively, on gated populations of CD4⫹ or
CD8⫹ T cells. Vertical bars indicate isotype controls. Experiments were
repeated three times.
plate-bound anti-CD3, and then transferred the cells to fresh plates
coated with additional anti-CD3 in combination with PK18 or rat
IgG. Surprisingly, PK18 was able to inhibit CD4⫹ T cell proliferation even after an initial overnight activation with anti-CD3,
similar to the inhibition observed for cells exposed to PK18 for the
entire culture period (Fig. 7).
Discussion
We have generated a panel of mAb specific for BTLA, a recently
discovered member of the Ig superfamily of cell surface proteins
(4). Only one of these Abs, PK18, was found to act as an agonist
for this coinhibitory molecule, thereby inhibiting facets of T cell
activation.
We were able to take advantage of the fact that PK18 preferentially binds the allele expressed in B6 mice, but not that in
BALB mice (3), to perform initial epitope-mapping studies. Binding of PK18 to BTLA.2, but not BTLA.1, is critically dependent
on residue 41, because the P41E substitution completely abolished
Ab binding. Other Abs, for example, PK3, bound to the position 41
mutant at wt levels, indicating that the inability of PK18 to bind the
mutant was not caused by a lack of surface expression. In addition,
the binding of PK3 to mutant 41 indicates that other epitopes on
the protein were not grossly distorted. The replacement of the proline at this position with a glutamic acid is likely to alter the local
structure of the protein, but might also prevent Ab binding due to
the addition of the negative charge.
Of the nine additional BTLA.2 mutants (encompassing 10 aa
changes), only two, the substitution at position 85 and the double
substitution at positions 45 and 47, had any effect on binding of
PK18. Another allele-specific mAb, PJ196, which lacks inhibitory
activity, was also affected to varying degrees by substitutions at
these same positions. Substitution at 85 had a small effect on binding of PJ196, whereas mutants 41 and 45/47 had significantly
greater effects on binding. Overall, however, the rank order of
binding of PK18 and PJ196 to these mutants differed (for PK18
mutants, 41⬍45/47 and 85; for PJ196, 45/47⬍41⬍⬍85), consistent
with overlapping, but distinct, epitopes recognized by the two Abs.
Given that PJ196 is a better binder to cells than PK18 (data not
shown), it seems likely that the dramatic difference in biological
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FIGURE 3. PK18-mediated inhibition does not involve induction of apoptosis or activation of Treg. A, T cells were activated in the presence or the
absence of PK18 as described in Fig. 1. Apoptosis was measured after 6 h
by propidium iodide/annexin V staining. As controls, apoptosis was also
analyzed in the presence of dexamethasone or PMA and ionomycin. Bars
represent the mean and SD of three independent experiments. B, Sorted
CD4⫹CD25⫺ T cells were activated by anti-CD3 and rat IgG (f) or antiCD3 and PK18 (䡺), and resulting proliferation was assessed as described
in Fig. 1. Results are representative of three experiments.
The Journal of Immunology
activity between these two mAb is due to the relatively subtle
differences in the epitopes bound.
These data should be helpful in producing and screening for
additional Abs that have agonist function. In an Ig fold, residues
41, 45, and 47 would be predicted to be on a solvent-exposed face
of the protein (data not shown). Whether this region of BTLA is
also involved in binding to HVEM, and whether there could be
allelic differences in that binding, remain to be determined. Interestingly, two BTLA.2-specific Abs generated by Murphy et al. (5)
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FIGURE 5. BTLA engagement inhibits IL-2 secretion. A, Supernatants
were collected 16 h after stimulation by anti-CD3 in the presence of rat IgG
or PK18 (as described in Fig. 2) and were analyzed for IL-2 secretion.
B, T cells were stimulated in the presence or the absence of PK18, as
described in A, with the addition in some cultures of the indicated concentration of recombinant murine IL-2. Proliferation was assessed as described
in Fig. 1. C, T cells were stimulated with anti-CD3 and rat IgG (u) or
anti-CD3 and PK18 (bold lines) with the addition of recombinant murine
IL-2 (800 pg/ml, high IL-2; 80 pg/ml, low IL-2). On day 2 of culture, cells
were harvested, and gated populations of CD4⫹ and CD8⫹ T cells were
analyzed for the expression of CD25 by flow cytometry. One representative
experiment of three is shown.
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FIGURE 6. Effects of costimulation on BTLA engagement. A, T cells
were activated by anti-CD3 and rat IgG (Œ) or anti-CD3 and PK18 (‚) in
the presence of anti-CD28, and proliferation was analyzed as described in
Fig. 1. B, CFSE-labeled T cells were stimulated as described in Fig. 2 with
anti-CD3 and rat IgG (u) or anti-CD3 and PK18 (f) in the presence of
anti-CD28. On day 3, cells were stained for CD4 or CD8 and were analyzed by flow cytometry. C, T cells were cultured with anti-CD3 and rat
IgG (u) or anti-CD3 and PK18 (bold lines) in the presence of anti-CD28.
On day 2 of culture, cells were harvested, and gated populations of CD4⫹
and CD8⫹ T cells were analyzed for the expression of CD25 by flow
cytometry. D, IL-2 secretion was assayed in the supernatants of overnight
cultures activated as described in B. E, Proliferation of AND TCR-Tg
CD4⫹ T cells cultured in the presence of BALB.K APCs and specific
peptide Ag with the indicated amount of soluble PK18 (䡺) or control rat
IgG (f) was measured by [3H]thymidine uptake as described in Fig. 1A.
Each graph represents one representative experiment of three.
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FUNCTIONAL ANALYSIS OF BTLA ENGAGEMENT ON T CELLS
FIGURE 7. Proliferation in preactivated T cells can be blocked by a
secondary BTLA signal. CD4⫹ T cells isolated from B6 mice were stimulated overnight with a titration of anti-CD3 and transferred to a new plate
coated with the indicated concentrations of anti-CD3 and rat IgG (F) or
anti-CD3 and PK18 (E). As controls, untransferred CD4⫹ T cells cultured
in the presence of anti-CD3 and rat IgG (f) or anti-CD3 and PK18 (䡺) are
shown. Data are displayed as the mean [3H]thymidine incorporation of
triplicate cultures (⫾SD) minus the cpm of T cells cultured alone. Two
independent experiments gave similar results.
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appear to recognize epitopes distinct from that of PK18 or PJ196,
although substitution at position 85 (our numbering) does eliminate binding of one of their Abs.
When assessed by uptake of thymidine, PK18 was found to be
a potent inhibitor of T cell proliferation. However, single-cell analysis of cell division by CFSE staining revealed that CD4⫹ T cells
were more susceptible to the inhibitory effects of PK18 than were
CD8⫹ T cells. Similarly, PK18 also inhibited CD25 up-regulation
on both CD4⫹ and CD8⫹ T cells, but the inhibition was again
more pronounced on CD4⫹ T cells. Resting CD4⫹ T cells express
modestly higher levels of BTLA than do CD8⫹ T cells (3) (Fig.
2B). Moreover, as shown in this study, BTLA is up-regulated with
faster kinetics on activated CD4⫹ compared with CD8⫹ T cells.
Thus, we cannot rule out that differences in expression may be one
factor in the increased susceptibility of CD4⫹ T cells to PK18mediated inhibition. Alternatively, CD8⫹ T cells may be intrinsically less sensitive to the inhibitory effects of PK18, especially
during their initial phase of proliferation, which may be IL-2 independent (14). We have seen similar results using a soluble form
of HVEM (data not shown); thus, it is unlikely that these results
are peculiar to the PK18 mAb.
Soluble PK18 also inhibited the proliferation of CD4⫹ T cells at
limiting concentrations of plate-bound anti-CD3 (Fig. 1B). This
was somewhat surprising, because it was previously reported that
BTLA phosphorylation, and thus presumably signaling, requires
co-cross-linking with the TCR (4). In these latter experiments, T
cell hybridomas overexpressing a Myc-tagged version of BTLA
lacking the external domain were treated with anti-Myc and antiCD3 Abs and cross-linked with a cross-reactive secondary Ab.
Whether the lack of the BTLA external domain or the use of hybridoma cells affected the requirement for co-cross-linking in this
system is not known. A soluble recombinant form of the BTLA
receptor HVEM, however, was also reported to inhibit T cell proliferation induced by limiting concentrations of plate-bound antiCD3 Ab, similar to the activity of PK18 (15, 16). Thus, PK18
appears to be a good mimic of the BTLA ligand.
We have found that BTLA engagement by PK18 is a potent
inhibitor of IL-2 secretion. However, the addition of exogenous
IL-2 at a concentration that is normally produced in these cultures
was not able to rescue proliferation or CD25 expression on T cells
activated in the presence of PK18. Thus, BTLA engagement has
additional IL-2-independent inhibitory effects on T cell activation.
In contrast to the effects on cytokine secretion, PK18 had no effect
on the induction of CD69, indicating that BTLA engagement does
not prevent initial activation of the T cell, but modifies the consequence of such activation. That BTLA might function as a response modifier is also supported by kinetic studies of susceptibility to a PK18-mediated inhibitory signal (see below). Although
IL-2 is a potent mediator of initial T cell proliferation in vitro, IL-2
may play a role in vivo in subsequent phases of T cell clonal
expansion and as a development of effector function in addition to
its effects on Tregs (reviewed in Ref. 17).
CD28-mediated costimulation lowers the threshold of T cell activation (18), potently enhances IL-2 production (13, 19) and
IL-2R expression (20), and promotes progression through the cell
cycle (21). At limiting concentrations of anti-CD3, PK18 was inhibitory even in the presence of anti-CD28. CD28 costimulation
was able to counteract the inhibitory effects of PK18 on cell division in the presence of high concentrations of anti-CD3. Even under these conditions, however, IL-2 production was significantly
inhibited by PK18. These findings are consistent with the ability of
APC that coexpress HVEM and CD80 to inhibit the proliferation
of TCR-Tg CD4⫹ T cells in response to low, but not high, concentrations of antigenic peptide (6). Similarly, high concentrations
of PK18 can inhibit Ag-specific T cell responses (Fig. 6D). This
inhibitory effect was due to binding of PK18 to the T cell, because
we used APC that would not be targeted by PK18. Thus, BTLA
engagement under the limiting conditions of costimulation or TCR
activation that may occur in vivo may deliver an inhibitory signal.
In the presence of anti-CD3, -CD28, and -PK18, the remaining
IL-2 produced was probably sufficient to induce T cell proliferation to near control levels, because similar concentrations of
exogenously added IL-2 rescued the proliferation of T cells
activated in the presence of PK18. It is also possible that the
ability of CD28 costimulation to regulate progression through
the cell cycle in an IL-2-independent manner (22) plays a role
in counteracting the inhibitory effects of BTLA engagement on
cell division.
BTLA up-regulation occurs over several days after T cell
activation. CD28-mediated costimulation increased the frequency of cells that up-regulated BTLA, but did not alter these
kinetics. Because the production of cytokine is a relatively early
event, the ability of PK18 to modify this response suggests that
BTLA engagement on naive T cells before up-regulation of the
cell surface protein can still deliver an inhibitory signal. Nevertheless, up-regulation of BTLA in vivo may be an important
factor in making T cells more susceptible to BTLA-mediated
inhibition. Interestingly, the BTLA ligand HVEM, also expressed by naive T cells, is down-regulated with similar kinetics
as those of BTLA up-regulation (9) (data not shown). This
might suggest that a BTLA-HVEM interaction is unlikely to
autonomously regulate T cell function.
Because PK18 failed to block CD69 up-regulation, we asked
whether BTLA engagement on preactivated CD4⫹ T cells would
still be inhibitory. Surprisingly, cell proliferation was inhibited
when CD4⫹ T cells were exposed to PK18 as late as 16 h after
initial culture with anti-CD3. Intravital imaging has recently
shown that during the first hours of initial CD4⫹ T cell activation,
serial short-lived interactions with APC bearing cognate peptide
are sufficient to up-regulate CD69 (23). Subsequent phases associated with more stable T cell-APC contacts appear to be required
for induction of cytokine and subsequent cell division. The fact
that inhibitory signals mediated by BTLA can be temporally separated from initial TCR activation raises the possibility that BTLA
engagement during this secondary phase of T cell-APC interaction
could modify the outcome of the T cell response.
The Journal of Immunology
Acknowledgments
We thank Parinaz Aliahmad for critically reading this manuscript, and
Alan Saluk, Cheryl Kim, and Kurt Van Gust for excellent technical assistance with cell sorting.
Disclosures
The authors have no financial conflict of interest.
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