| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Transplantation Biology Group, Medical Research Council Clinical Sciences Centre, and
Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Molecular dissection of minor H antigenic loci has revealed the
presence of both MHC class I- and class II-restricted epitopes,
indicating a potential requirement for cooperation between the
CD4+ and CD8+ T cell
subsets to generate effective minor H-specific responses
(3). Indeed, presentation of a single MHC class
I-restricted self epitope in the absence of CD4+
T cell help or depletion of either the CD4+ or
the CD8+ recipient T cell subset can lead to the
induction of nonresponsiveness (4, 5). Recently, the
mechanism of CD4+ T cell help has been defined as
operating through the presenting dendritic cell (DC), equipping the APC
to deliver activating signals to subsequently encountered
CD8+ T cells (6, 7, 8). The HY minor H
Ag is particularly amenable to analysis because all the epitopes are
encoded by genes on the Y chromosome, and in mice, these map to the
Sxr deletion interval (
Sxr) (9).
In inbred mouse strains, females grafted with syngeneic male skin can
respond only to products of these genes. Some of these Y chromosome
genes have X chromosome homologues encoding related peptides. It is the
processing and selection of X and Y chromosome-encoded peptides by MHC
Ag-presenting molecules that, in part, determines the magnitude of the
HY response. In mice, responsiveness to HY varies widely between
strains; females of H2b haplotype strains are
strong responders capable of rejecting primary syngeneic male skin
grafts. In contrast, females of some H2k strains
can reject syngeneic male grafts following immunization, whereas
females of most H2d strains are nonresponders to
HY (10). The basis for low reactivity to HY in
non-H2b strains is not entirely clear because
F1 females with one H2b
parent can reject male skin grafts of the non-H2b
parent. Furthermore, after footpad (FP) immunization with syngeneic
male cells, some low responder strains can generate
CD4+ and CD8+ HY-specific T
cells (11).
Recently, MHC class I- and II-restricted HY peptide epitopes of the H2b haplotype have been identified (12, 13, 14). The two MHC class I-restricted peptides, WMHHNMLDI and KCSRNRQYL, originate, respectively, from the Uty and Smcy genes and associate with the H2-Db molecule. Some evidence suggests that the WMHHNMLDI peptide is immunodominant because T cells with specificity for H2-Db/KCSRNRQYL are less commonly isolated than those with the alternate specificity (15). Further, transgenic mice expressing a TCR specific for the KCSRNRQYL epitope fail to reject skin grafts, suggesting that this receptor may have poor reactivity for its ligand (16). TCR-transgenic CD8+ T cells from this strain also have an apparent defect in cytotoxicity in response to male targets, although this can be overcome by provision of an elevated density of the cognate peptide (17, 18).
To further characterize the immune response to HY in the context of the strong responder H2b haplotype, we have produced tetrameric H2-Db complexed with the two peptides. Tetrameric MHC class I/peptide complexes have been used extensively to detect and analyze Ag-specific T cells during viral infections and for the detection of tumor-specific and autoreactive T cells (19, 20, 21, 22, 23, 24, 25, 26). They have also been applied in one study for the detection of minor H-specific cells potentially involved in GvH disease following bone marrow (BM) transplantation (27). We were interested in comparing the efficacy of different cell populations for the stimulation of epitope-specific HY responses and in determining the extent of clonal expansion in HvG responses in comparison to those seen during acute viral infection. We reasoned that tetramers would also be able to detect cross-primed CD8+ T cells if this mode of Ag presentation was effective for the HY Ag.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Soluble MHC/peptide tetramers were produced using a modified
version of that described by Altman et al. (19).
Recombinant MHC class I H2-Db H chain containing
a 15-aa C-terminal substrate peptide (biotin-specific peptide)
for BirA-dependent biotinylation was expressed in BL21 E.
coli. Protein expression was induced at mid-log growth phase with
0.5 mM isopropyl-
-D-thiogalactosidase (Sigma,
St. Louis, MO). After 5 h, cells were harvested and lysed by
overnight freeze/thaw (-70°C). H chain protein was purified from
inclusion bodies and denatured in 8 M urea. Human
2-microglobulin (2m)
was prepared in the same way.
H chain and 2m were refolded in 1 liter of
arginine buffer (100 mM Tris (pH 8), 400 mM L-arginine, 5
mM reduced glutathione, 0.5 mM oxidized glutathione, and 2 mM EDTA)
with the HY peptides WMHHNMLDI or KCSRNRQYL at a molar ratio of
1:2:10, respectively, for 48 h at 4°C. Refolded material was
concentrated in a N2-pressurized stir cell to 
5 ml
and was purified by gel filtration on a Sephadex G-75 column (Amersham
Pharmacia Biotech, Piscataway, NJ). The complex was biotinylated using
BirA (Avidity, Denver, CO) for 12 h at 25°C (reaction
conditions: 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM biotin D, 5 mM ATP,
and 5 mM MgCl2). Biotinylated complexes were
purified by gel filtration as described previously, followed by Mono Q
ion exchange (Amersham Pharmacia Biotech). Tetramers were prepared by
mixing the biotinylated complex with PE-labeled ExtrAvidin-R-PE
conjugate (Sigma) at a molar ratio of 4:1.
Cell culture
Cell lines and clones were cultured in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 10% FCS (Labtech International, Sussex, U.K.), 100 U/ml each penicillin and streptomycin, 50 mM 2-ME, 2 mM glutamine, and 10 mM HEPES in 2-ml Linbro 24-well plates (ICN Pharmaceuticals, Costa Mesa, CA ) at 37°C in 5% CO2. Bulk cultures from mouse spleen were restimulated every 10 days with 5 x 106 irradiated syngeneic splenic APCs and 20 IU rIL-2/ml. The HY Db/Uty-specific T cell clone CTL-10 (28) was restimulated every 14–21 days. RMA-S is a transporter-deficient H2b thymoma cell line.
Cytotoxicity assays
Fresh or cultured cells were assayed 5 days following stimulation with Ag and 20 IU/ml rIL-2. RMA-S target cells were labeled with 51Cr alone or in the presence of 1 mM peptide for 1 h at 37°C, washed three times, and plated at 104 cells/well in round-bottom 96-well plates. Effector cells were incubated with the target cells at E:T ratios of 10:1, 3:1, and 1:1. After 4 h, 100 µl of supernatant was collected and 51Cr release was measured using a gamma counter. Results were calculated at an E:T ratio of 10:1. The percentage of lysis was calculated from the formula 100 x (E - M)/(T - M), where E is the experimental release, M is the spontaneous release in the presence of medium alone, and T is the maximum release in the presence of 5% Triton X-100.
Flow cytometry
Spleen cell suspensions were depleted of B cells with sheep anti-mouse Ig Dynabeads (Dynal, Wirral, U.K.). Aliquots of 106 cells were stained in 50 µl of PBS containing 2% FCS (FACS buffer) with 1 µl of HY Db tetramer for 10 min at room temperature, and then with FITC- or PerCP-labeled anti-CD8 Ab (BD PharMingen, San Diego, CA) for 15 min at 4°C, followed by two washes in FACS buffer. Ag-specific cells were phenotyped with FITC-labeled anti-CD69, -CD44, -CD45RB, and -CD62 ligand Abs (BD PharMingen). DCs were phenotyped with FITC-labeled anti-CD11c and -B7-1 and biotinylated anti-H2-Ab and -B7-1; biotinylated Abs were detected with streptavidin-PerCP (BD PharMingen). To detect apoptosis, cells were first stained with tetramer as previously described, stimulated with HY peptide-pulsed APCs, and stained with FITC-conjugated annexin V (BD PharMingen). Samples were acquired on either a FACScan or FACSCalibur instrument (BD Biosciences, San Jose, CA). Data were analyzed using CellQuest software (BD Biosciences).
CFSE labeling
Cells were washed and resuspended at a concentration of 107/ml in PBS. CFSE (Molecular Probes, Eugene, OR) (29) was added at a final concentration of 5 mM, and cells were incubated at 37°C for 10–15 min. Cells were washed three times before restimulation with male APCs.
Stabilization of H2-Db on RMA-S
RMA-S cells (106) were incubated with serial 10-fold dilutions of either Db//WMHHNMLDI or Db/KCSRNRQYL in 200 µl of RPMI 1640 at 26°C overnight. After washing, they were incubated at 37°C for 1 h to allow the decay of empty class I molecules and were the stained with FITC-labeled B22.249 (anti-H2-Db).
Immunization and skin grafting
Female 4- to 8-wk-old C57BL/6 mice (Olac, Bicester, U.K.) were
used as responders. The
2m-/- (C57BL/6
background) mice were obtained from The Jackson Laboratory (Bar Harbor,
ME). Wild-type or 2m-deficient male C57BL/6
tissues were used as immunogen. DCs were prepared from BM cultured with
GM-CSF for 7 days. To promote maturation, cultures were passaged
24 h before harvesting (30). The majority of cells in
these cultures were CD11c positive, and of these >90% were MHC class
II and B7-1 positive, but only a minority were B7-2 positive (data not
shown). For the time-course experiments, 106 DCs
were injected s.c. into FPs. Grafting of tail skin was conducted using
the method of Billingham et al. (31). Other immunizations
were conducted with 5 x 106 DCs or
single-cell suspensions of fresh BM or spleen. For peptide
immunizations, cells were incubated with the appropriate peptides (10
µM) at 37°C for 90 min, washed twice with PBS, and 5 x
106 cells were injected i.p. in 200 µl of
PBS.
ELISPOT
MultiScreen-IP plates (96-well; Millipore, Bedford, MA) were
coated with 5 µg/ml anti-mouse IFN-
mAb (BD PharMingen),
blocked with RPMI 1640–10% FCS, and washed with PBS. Peptide-pulsed
stimulator cells were prepared by incubating 107
irradiated female spleen cells in 10 µM peptide for 2 h at
37°C. Peptide-pulsed or unpulsed spleen cells (2 x
105) were added to the wells with
105 B cell-depleted responder spleen cells to
give a 200-µl final volume. After a 24-h incubation, cells were
water-lysed, and the plates washed were washed six times with PBS and
0.1% Tween 20. A total of 100 µl 20 ng/ml biotinylated
anti-mouse IFN- mAb in PBS and 1% BSA was added and incubated
overnight at 4°C. Plates were washed as described above, and 1 µg
of streptavidin-alkaline phosphatase (Sigma) was added in 100 µl of
PBS and 1% BSA. After 1 h at room temperature, the plates were
washed four times as described above and two times with 0.1 M Tris (pH
7). Plates were developed by adding 5-bromo-4-chloro-3-indolyl
phosphate/nitroblue tetrazolium substrate (Sigma).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The specificity of the H2-Db tetramers was
confirmed by staining Ag-specific T cells. The
Db/WMHHNMDLI tetramer preparation stained
virtually 100% of the T cell clone CTL10 (28), which is
specific for the Db/WMHHNMDLI complex, whereas
this T cell clone was not stained by the
Db/KCSRNRQYL tetramer (Fig. 1
). The converse result was obtained
using spleen cells from females of the B6.2.16
(Db/KCSRNRQYL specific) TCR-transgenic strain
(32). In this case, the CD8+ T cells
were stained specifically only with the
Db/KCSRNRQYL tetramer preparation. These data
confirm the ability of the tetramer preparations to stain the
appropriate T cell population and to distinguish between TCRs reactive
to the same MHC molecule but associated with distinct peptides.
|
A time-course experiment was undertaken to establish the kinetics
and extent of HY Db-specific
CD8+ T cell expansion in female
H2b mice following immunization with male DCs.
DCs were produced by 7 days of GM-CSF stimulation of BM. Female
recipients were immunized in the FP with 106 male
or female DCs and spleen cells analyzed at 7, 14, 21, 35, and 76 days
for tetramer-positive CD8+ cells. Exposure to
female skin grafts or DCs did not lead to expansion of the
Db/HY tetramer-reactive populations, which
appeared as a background of 0.1% of the splenic
CD8+ T cell population in both untreated mice and
those given female cells (Fig. 2, A and B). Males are tolerant to HY principally
through central thymic deletion of reactive cells; we did not observe
any difference in Db/HY tetramer-positive cell
frequencies in CD8+-single-positive
thymocytes/spleen cells between male and female (data not shown). This
is expected, because the frequency of HY-specific
CD8+ T cells in naive females was anticipated to
be very low.
|
, C–F. Three points emerge from the
accumulated data on ex vivo staining of spleen cells following DC
immunization (Fig. 3A).
Firstly, the proportion of either HY-specific
CD8+ T cell cohort generally does not expand
above 1% of the total splenic CD8+ population.
Secondly, there is variation between individual responders both in the
size and skew of the two cohorts with no clear immunodominance.
Thirdly, the response is maintained at approximately the same level
throughout the observation period (76 days).
|
Primary syngeneic male skin grafts are rejected by female
H2b mice with median survival time of 35 days
in comparison with the faster response (approximate median survival
time of 15 days) following immunization (9). To establish
whether immunization with male DCs and its influence on the speed of
graft rejection would be reflected in an increase in the
Db/HY-specific CD8+ T cell
cohorts detected, female recipients were given male or female skin
grafts 2 wk after male DC immunization, leading to graft rejection at
around days 13–15. Fig. 3B shows the appearance of the
HY-specific cohorts detected during the second-set skin-graft-rejection
response in a series of recipients. Firstly, as was seen following DC
immunization alone, variation between individual responders is observed
in both the magnitude and fine specificity of the response; however, no
evidence of significant skew toward the immunodominant epitope was
observed. Secondly, the magnitude of the response is, in many cases,
above that seen following DC immunization alone. However, the peak
expansion size seen for either specificity is 2% of the total
splenic CD8+ T cell subset.
HY responses in mice chronically exposed to male tissue
Because no clear evidence of epitope immunodominance emerged in
the above experiments using standard immunization procedures, we used a
protocol designed to more extensively stimulate the anti-HY immune
response and promote clonal selection, which might reveal epitope
dominance. Female H2b recipients were immunized
with male spleen cells, and 18 days later, the mice were sublethally
irradiated (400 rad) and reconstituted with male BM. Five weeks later,
male skin grafts were applied. Recipients that rejected both their BM
and skin graft, and hence showed evidence of a vigorous response to HY,
were analyzed with tetramers 5–13 days following rejection of skin
(Fig. 3C). This chronic stimulation led to considerably
larger expansions of HY-specific CD8+ T cells;
interestingly, the majority of mice (9 of 10) showed marked
preferential expansion of the immunodominant
Db/Uty-specific cells (3- to 18-fold)
over the alternate specificity.
Influence of cell type and route of immunization
DCs are considered to be critical APCs for the initiation of an
immune response. Ags that are present within other cell types can
access host DCs via endocytosis and can be processed and presented to T
cells via the indirect or cross-priming route (33). We
have directly compared the efficacy of male spleen, BM, and DCs to
stimulate a class I-restricted HY response. Injection of 5 x
106 cells i.p. led to the detection of
HY-specific CD8+ T cells 14 days later;
surprisingly, DCs were inferior to spleen or BM (Fig. 4A). BM was the most effective
immunizing tissue, leading to 6, 10, and 23% of the
CD8+ compartment becoming HY specific in three
recipients (Db/Smcy and
Db/Uty populations). The efficacy of
spleen and BM is dependent on the cell dose used because DC
immunizations of 5 x 105 and 5 x
106 promoted similar expansions of
tetramer-positive cells, whereas spleen or BM cell immunizations of
5 x 105 led to small (<0.5%) cohorts of
tetramer-positive cells (data not shown).
|
B shows representative data from each group of three
recipients. The i.v. and i.p. routes gave similar levels of cell
expansion, whereas FP immunization was markedly less effective.
Functional CD8+ cytotoxic T cell responses to
minor H Ags require help provided through activation of
CD4+ T cells. To confirm that help is necessary
for CD8+ T cell expansion in vivo, we used B10.GD
(H2-Kd,-Ad,and
-Db) mice that lack the HY-presenting class II
molecule Ab and are HY nonresponders. Female
B10.GD recipients receiving male B10.GD BM did not show expansion of
either HY CD8+ T cell population (data not
shown), although they did display the appropriate MHC class I/peptide
complexes.
Uty- and Smcy-derived peptides bind to and
stabilize empty Db molecules on the surface of
RMA-S cells efficiently, although at the more physiologically relevant
lower peptide concentration range, Uty stabilizes
Db more efficiently (Fig. 5A). To test whether female
APCs coated with HY peptides are able to reproduce the immunization
process, female spleen cells or DCs were coated with the
H2b HY peptide epitope set including the
H2-Ab-restricted epitope NAGFNSNRANSSRSS
(14). Cells (5 x 106) were
injected i.p., and spleen cell populations were collected after 14
days. Peptide-coated cells failed to induce T cell cohorts detectable
by tetramer staining whether the MHC class I-restricted peptides were
present individually or together and in the absence or presence of the
Ab peptide (Fig. 5B and data not
shown). Furthermore, tetramer-positive cells were not seen following
further in vitro stimulation of spleen cells, confirming the inability
of peptide-coated cells to immunize effectively (Fig. 5C).
|
2m-deficient male spleen cells as immunogen.
This material stimulated the expansion of tetramer-positive
CD8+ T cohorts of both specificities, although
the populations were 10% of those generated by class I-sufficient
spleen cells, suggesting that indirect presentation, although clearly
operative, is not the principal pathway for this immune response (Fig. 5, E and F). The cross-primed cells expanded
further in vitro following exposure to Ag, as seen for directly primed
cells (data not shown). As observed for the direct pathway of Ag
presentation, we did not see immunodominance during this period of the
HY immune response, suggesting that both epitopes are presented via the
indirect pathway with similar efficacy. To confirm that the HY helper
response was not compromised in the absence of MHC class I, an ELISPOT
assay was used to look at priming of T cells specific for the class II
Ab-restricted HY epitope and showed the response
to be similar with MHC class I-sufficient or -deficient immunogen (Fig. 5D). Phenotype of HY-specific CD8+ T cells
Spleen cells from female mice immunized with male DCs or DCs followed by a male skin graft were analyzed ex vivo at day 28 by dual staining with MHC peptide tetramer and Abs specific for markers of activation. The phenotype of both tetramer-positive populations was CD69lowCD44highCD45RBhighCD25lowCD62 ligandlow (data not shown), which is typical of Ag-experienced cells and may represent a recirculating and/or memory pool.
In vitro cultures
In all cases in which Ag-specific populations of
CD8+ splenic T cells were detected ex vivo with
either tetramer, expansion of the corresponding population was seen
following in vitro stimulation with male APC; importantly, this was not
seen with spleen cells from unimmunized mice (data not shown). The
correlation between ex vivo detection by tetramer and in vitro
expansion is important with regard to the identity of the small
populations of CD8+ T cells staining with
Db/HY tetramers in nonimmunized animals. These
cells constitute 0.1% of the CD8+ population
but do not form a clustered cohort in the FACS analysis and are
distributed throughout the quadrant (Fig. 2, A and
B).
In vivo, because immunodominance was apparent only following extensive
stimulation with male cells, it was of interest to determine whether
immunodominance may be more apparent following in vitro stimulation.
Primary splenic cultures from mice immunized with male DCs followed by
skin grafting and which, at the time of harvest, contain similar
proportions of both HY CD8+ T cell specificities
were restimulated in vitro. The relative expansion of the two cell
populations within the same culture can be assessed during in vitro
propagation. In cultures of this type, the
Db/WMHHNMDLI-specific population was found to
preferentially expand in comparison to the
Db/KCSRNRQYL population. A representative example
is shown in Fig. 6, illustrating the
dominance of the Db/WMHHNMDLI-specific population
following two in vitro restimulations. The mechanism of in vitro
dominance may involve a more rapid expansion of the
Db/WMHHNMDLI-specific cells; alternatively, the
Db/KCSRNRQYL-specific population may be
undergoing activation-induced cell death at a greater rate. To
investigate these possibilities, T cell cultures containing both
specificities were stained with CFSE and cultured for an additional 5
days to determine the relative extent of cell division. Annexin V
staining was used to gauge the degree of apoptosis. The result of the
CFSE stain indicates that, within these cocultures, the immunodominant
Db/WMHHNMDLI-specific population has undergone
more cell division than the Db/KCSRNRQYL-specific
population, consistent with an in vitro growth advantage (Fig. 7, A and C).
Furthermore, a larger fraction of
Db/KCSRNRQYL-specific cells consistently stained
with annexin V (Fig. 7, B and D).
|
|
A, the cytotoxic effector
function of both populations was directed predominantly at the cognate
peptide-loaded RMA-S target cells. The low level of cytotoxicity
directed at the noncognate peptide-loaded target is attributable to
residual T cell populations of the alternate specificity. It was also
possible to clone directly from spleens of immunized mice. Although at
the time of cloning both specificities were present at similar
frequencies, Db/KCSRNRQYL clones were
under-represented (Expt. 1, 1 of 23; and Expt. 2, 3 of 32), consistent
with the more robust in vitro growth of the
Db/WMHHNMDLI-specific cells. Because all 54
clones stained with one or the other tetramer, we find no evidence for
additional antigenic complexity. The poor representation of the
Smcy-specific clones suggests that their in vitro endurance
relative to the Uty-specific cells is, in part, cell
autonomous and not due to coculture. Assessment of the clones by
cytotoxicity revealed complete correlation between tetramer stain and
function (Fig. 8B). Within the panel of
Db/WMHHNMDLI-specific clones isolated following a
primary HY response, some had markedly weaker staining with tetramer,
suggesting that a range of TCR/MHC-peptide affinities are expanded by
antigenic stimulation (Fig. 8C), although this was not seen
in the clones isolated from animals exposed to male DC and skin
graft.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this study, we wished to define the kinetics and extent of T cell expansion in response to primary immunization with male DC and other APC types, following primary- and second-set skin graft rejection and in chronically stimulated anti-HY responses. Responses to the two defined MHC class I-presented HY epitopes of the H2b haplotype have been compared using specific Db tetramers. We were also particularly interested in gaining insight into the mechanism underlying the reported immunodominance of the Db/WMHHNMDLI epitope over the Db/KCSRNRQYL epitope. Following immunization of female mice with male DCs, spleen, or BM, Ag-specific T cells were detectable in spleen with both tetramers from day 14. Interestingly, immunization with primary skin grafts was less effective than DCs in stimulating the MHC class I HY response, perhaps reflecting the relative numbers of APCs involved in each immunization protocol. Furthermore, immunization with DCs is likely to result in systemic immunization involving multiple lymph nodes, whereas a skin graft is likely to initially prime an immune response only in the draining lymph nodes. Skin grafting subsequent to male DC immunization again led to the appearance of CD8+ T cells of both specificities. In comparison to DC immunization alone, further immunization by exposure to male grafts led to a small elevation of the Ag-specific CD8+ T cell frequency, although the responses to the individual epitopes did not exceed 2%. At a high cell number (5 x 106), spleen and BM were found to be more efficient than BM-derived DCs at stimulating tetramer-positive T cell expansion. At this high cell number, spleen and BM may contain sufficient DCs to effectively prime the HY response and semiprofessional APC populations to effectively expand the primed cells. Strikingly, none of these immunization protocols led to immunodominance of the Uty epitope over the Smcy epitope. Because skewing was not apparent during primary and secondary HY responses, it is likely that the precursor frequencies of CD8+ T cells specific for the two class I epitopes do not differ markedly. To test whether immunodominance would arise following persistent Ag exposure, mice were repeatedly immunized with male tissue. In contrast to the conventional immunization protocols, mice immunized with male spleen cells and which subsequently rejected male BM and skin grafts showed a response intensely skewed to the immunodominant epitope. Because skewing was only apparent following chronic antigenic stimulation, it is clear that the typical anti-HY response involves both Db-restricted epitopes in similar measure.
These observations make it unlikely that lack of peptide availability
or TCR affinity can simply explain the immunodominance that emerges
with persistent stimulation; indeed, tetramer staining of the
Db/Smcy population was as bright as
for the Db/Uty population. Rather, the
evolution of the immune response during continuing stimulation shapes
the outcome. It is not clear why, as populations, the
Db/Smcy-specific cells are ultimately
less successful than the
Db/Uty-specific cells when the immune
response is more intensely stimulated. Wolpert et al. (34)
and Pion et al. (35) have suggested that immunodominance
can emerge due to interference between responding T cells because
expression of epitopes on the same APC was required for immunodominance
to emerge. Interestingly, as reported here, competition for CTL
survival in mixed in vitro culture was also seen for B6 anti-BALB.B
minor responses and required coexpression of epitopes on the APC. The T
cell cloning experiments described here are consistent with cell
survival in vitro being, at least in part, cell autonomous and not
dependent on competition or cross-talk between populations.
Db/Uty is immunorecessive to the
B6(dom1) epitope (35); the dramatic difference in
cell surface density of the B6(dom1) peptide, present at around 1000
copies/APC, in comparison to Db/Uty,
which is present at around 10 copies/APC, has been proposed, in part,
to explain the hierarchy of immunodominance (36). We find
that the response to Db/Uty is, under
chronic stimulation, dominant to that of
Db/Smcy, suggesting that a high
peptide density is not required, at least for the limited
immunodominance described here. Relatively small differences in
proliferative or survival signals delivered by APCs may cumulatively
account for strong skewing during prolonged stimulation. For example,
the greater affinity of the Db/Uty
interaction (Fig. 5) may give this combination a marginal advantage
over the immunorecessive epitope combination. It may be important
during the early stages of an immune response to use the full range of
T cells available for an epitope-specific response, and only when cell
numbers have expanded are the less effective cells no longer useful for
the response. Consistent with this notion, the clonal analysis (Fig. 8C) detected lower affinity TCRs only in the primary
response. A study using tetramers to look at immunodominance within the
CD8+ response to viral epitopes in rhesus monkeys
chronically infected with simian/HIV rarely found significant cell
populations specific for the immunorecessive epitope (37).
It will be of interest to determine how rapidly immunodominance emerges
for other chronic immune responses.
However, immunodominance was very apparent during short-term in vitro culture, as the Db/WMHHNMDLI-specific T cells rapidly dominated the cultures. The immunodominance we see in vitro can explain the deficit of T cell clones with Db/KCSRNRQYL specificity, because these will generally be isolated from cultures expanded in vitro before cloning. Why should immunodominance be more apparent in vitro than in vivo? One possibility is that T cell proliferation is less regulated in the absence of the multiple highly regulated mechanisms that operate in the in vivo microenvironment to self-limit T cell expansion, including Fas/Fas ligand interaction (38), CTLA-4/B7 interaction (39), and IL-2-regulated apoptosis (40). Relatively small differences in access to APC surface due to variation in TCR affinity or half-life may quickly translate to rapid outgrowth of one population in vitro.
The data presented here show that in robust HY responder
H2b mice, expansions of
CD8+ T cells specific for individual HY epitopes
during skin graft rejection or following immunization are substantial,
although of smaller magnitude (<10%), than the extremes seen during
some acute viral infections. The specific cell density at the graft
site may, of course, be higher. It is likely that viral replication
leading to an increasing Ag load combined with activation of aspects of
innate immunity following virus recognition contributes to the large T
cell expansions seen in some acute virus infections. However, chronic
stimulation involving multiple male APC types promoted large expansions
skewed to the immunodominant epitope. One critical observation was the
very extensive HY-specific CD8+ T cell expansions
seen following immunization with BM or spleen, even though the MHC
class I-restricted antigenic complexity of HY is very limited.
Importantly, spleen and BM populations contain only a small percentage
of professional APC types, such as DCs, and yet result in expansions of
the same order as in acute viral infection. Indeed, highly enriched
BM-derived DC populations were markedly less effective at stimulating
CD8+ T cell expansion; poor stimulation was not
due to their rapid disappearance, because BM-derived DCs of the type
used here persist for 3 wk in vivo (41).
Cross-priming following cytoplasmic degradation of Ag is an important
route for Ag presentation (33) and has been proposed to be
of particular relevance in transplantation (42). To test
whether the large expansions of HY-specific CD8+
T cells we see following spleen or BM immunization are stimulated
principally through direct or indirect Ag presentation, we used
2m-deficient male spleen cells and found the
direct route to predominate. We find the indirect route of Ag
presentation to account for 10% of the response and, hence, to play
a minor role in spleen-cell immunization. This may not be surprising
because spleen contains professional APCs as well as many
semiprofessional APCs. Because HY expression is ubiquitous, we will be
able to dissect the role of different cell types and routes of
immunization for entry into the indirect pathway. Ag presentation for
initiation of an immune response is DC mediated, whereas subsequent
expansion involves both semi- and nonprofessional APCs; again, we will
be able to dissect the capabilities of different donor cell types to
engage in these two stages via the indirect route. These data show that
large expansions of CD8+ T cells are not
restricted to acute infection and have direct relevance to strategies
for the development of protective CD8+ immunity
through vaccination and the study of HvG responses compromising BM
engraftment after transplantation.
The role of CD4+ T cell help in minor Ag responses is critical for the development of cytotoxic CD8+ cells (4, 43, 44) and for skin graft rejection (4, 5). In the absence of help, the CD8+ T cell compartment can be rendered tolerant. Female B10.GD (Db, Ad, and Db) mice do not reject male skin due to lack of the critical Ab-restricted helper response. In this strain, we did not see any tetramer-positive cells, suggesting that tolerance does not involve expansion of a regulatory CD8+ population. In view of this, it was not surprising that CD8+ T cell expansions were not seen following the use of MHC class I-restricted HY peptides coated onto female B6 DCs or spleen. However, additional provision of the HY Ab-restricted peptide did not lead to tetramer-positive CD8+ T cell expansions, although accelerated graft rejection was observed (data not shown), suggesting some HY-specific CD4+ and/or CD8+ T cell priming. The maturation state of the DCs, density and half-life of the peptide on the cell surface, and the potentially low efficacy of cross-priming with peptidic Ag relative to cellular Ag may contribute to the failure to see MHC class I tetramer staining. It will be of interest to directly determine, using MHC class II tetramers, the efficacy of the HY Ab peptide to expand the CD4+ compartment.
The immune response to MHC class I- and II-presented self peptides is of significant clinical importance in a number of contexts. Responses to minor H Ags following BM transplantation contribute to GvH and HvG responses in both MHC-matched and -mismatched situations, self peptides derived from differentiation Ags or mutant proteins are potential targets for tumor immunotherapy, and inappropriate responses to self peptides can result in autoimmunity. Depending on the context, manipulation of T cell responses to self peptides, leading to diminution or augmentation of a response, represents a powerful strategy for improving clinical outcome. Characterization of the normal course of the MHC class I-restricted response to the model minor H Ag HY provides a useful experimental model for the investigation of the mechanism(s) of action of interventions designed to suppress the immune response to transplanted tissue. This report shows how monitoring of the relevant population(s) can be undertaken both ex vivo and following further in vitro expansion.
|
Acknowledgments |
|---|
2m expression plasmids. We thank
Dr. Tessa Crompton for the 2m-deficient mice, Dr. Derry
Roopenian for the CTL-10 T cell clone, Dr. Alan Bennett for the kind
gift of BirA enzyme, and Dr. Hans Stauss for useful discussion and
comments on the manuscript. |
Footnotes |
|---|
2 Abbreviations used in this paper: H, histocompatibility; BM, bone marrow; DC, dendritic cell; GvH, graft-vs-host; HvG, host-vs-graft; 2m, 2-microglobulin.
Received for publication January 11, 2001. Accepted for publication July 30, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2M tetramers to quantitate CD8+ cytotoxic T lymphocytes specific for dominant and nondominant viral epitopes in simian-human immunodeficiency virus-infected rhesus monkeys. J. Virol. 73:5466.
T lymphocytes for apoptosis. Nature 353:858.[Medline]
This article has been cited by other articles:
|
|
 |
|
  W. F. N. Chan, H. Razavy, and C. C. Anderson Differential Susceptibility of Allogeneic Targets to Indirect CD4 Immunity Generates Split Tolerance J. Immunol., October 1, 2008; 181(7): 4603 - 4612. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Jusforgues-Saklani, M. Uhl, N. Blachere, F. Lemaitre, O. Lantz, P. Bousso, D. Braun, J. J. Moon, and M. L. Albert Antigen Persistence Is Required for Dendritic Cell Licensing and CD8+ T Cell Cross-Priming J. Immunol., September 1, 2008; 181(5): 3067 - 3076. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. F. Moxham, J. Karegli, R. E. Phillips, K. L. Brown, T. T. Tapmeier, R. Hangartner, S. H. Sacks, and W. Wong Homeostatic Proliferation of Lymphocytes Results in Augmented Memory-Like Function and Accelerated Allograft Rejection J. Immunol., March 15, 2008; 180(6): 3910 - 3918. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Verginis, K. A. McLaughlin, K. W. Wucherpfennig, H. von Boehmer, and I. Apostolou Induction of antigen-specific regulatory T cells in wild-type mice: Visualization and targets of suppression PNAS, March 4, 2008; 105(9): 3479 - 3484. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Furmanski, C. Ferreira, I. Bartok, S. Dimakou, J. Rice, F. K. Stevenson, M. M. Millrain, E. Simpson, and J. Dyson Public T Cell Receptor -Chains Are Not Advantaged during Positive Selection J. Immunol., January 15, 2008; 180(2): 1029 - 1039. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Weng, J. Dyson, and F. Dazzi Low-intensity transplant regimens facilitate recruitment of donor-specific regulatory T cells that promote hematopoietic engraftment PNAS, May 15, 2007; 104(20): 8415 - 8420. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Mirenda, S. J. Jarmin, R. David, J. Dyson, D. Scott, Y. Gu, R. I. Lechler, K. Okkenhaug, and F. M. Marelli-Berg Physiologic and aberrant regulation of memory T-cell trafficking by the costimulatory molecule CD28 Blood, April 1, 2007; 109(7): 2968 - 2977. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. J. Robertson, J.-G. Chai, M. Millrain, D. Scott, F. Hashim, E. Manktelow, F. Lemonnier, E. Simpson, and J. Dyson Natural Regulation of Immunity to Minor Histocompatibility Antigens J. Immunol., March 15, 2007; 178(6): 3558 - 3565. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Firat, L. Saveanu, P. Aichele, P. Staeheli, J. Huai, S. Gaedicke, A. Nil, G. Besin, B. Kanzler, P. van Endert, et al. The Role of Endoplasmic Reticulum-Associated Aminopeptidase 1 in Immunity to Infection and in Cross-Presentation J. Immunol., February 15, 2007; 178(4): 2241 - 2248. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ferreira, A. Furmanski, M. Millrain, I. Bartok, P. Guillaume, R. Lees, E. Simpson, H. R. MacDonald, and J. Dyson TCR-{alpha} CDR3 Loop Audition Regulates Positive Selection J. Immunol., August 15, 2006; 177(4): 2477 - 2485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Palmowski, U. Gileadi, M. Salio, A. Gallimore, M. Millrain, E. James, C. Addey, D. Scott, J. Dyson, E. Simpson, et al. Role of Immunoproteasomes in Cross-Presentation J. Immunol., July 15, 2006; 177(2): 983 - 990. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Millrain, D. Scott, C. Addey, H. Dewchand, P. Ellis, I. Ehrmann, M. Mitchell, P. Burgoyne, E. Simpson, and J. Dyson Identification of the Immunodominant HY H2-Dk Epitope and Evaluation of the Role of Direct and Indirect Antigen Presentation in HY Responses J. Immunol., December 1, 2005; 175(11): 7209 - 7217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Bouneaud, Z. Garcia, P. Kourilsky, and C. Pannetier Lineage relationships, homeostasis, and recall capacities of central- and effector-memory CD8 T cells in vivo J. Exp. Med., February 22, 2005; 201(4): 579 - 590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Maile, C. A. Siler, S. E. Kerry, K. E. Midkiff, E. J. Collins, and J. A. Frelinger Peripheral "CD8 Tuning" Dynamically Modulates the Size and Responsiveness of an Antigen-Specific T Cell Pool In Vivo J. Immunol., January 15, 2005; 174(2): 619 - 627. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. J. Fry, M. Sinha, M. Milliron, Y.-W. Chu, V. Kapoor, R. E. Gress, E. Thomas, and C. L. Mackall Flt3 ligand enhances thymic-dependent and thymic-independent immune reconstitution Blood, November 1, 2004; 104(9): 2794 - 2800. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Rice, S. Buchan, H. Dewchand, E. Simpson, and F. K. Stevenson DNA Fusion Vaccines Induce Targeted Epitope-Specific CTLs against Minor Histocompatibility Antigens from a Normal or Tolerized Repertoire J. Immunol., October 1, 2004; 173(7): 4492 - 4499. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-G. Chai, E. James, H. Dewchand, E. Simpson, and D. Scott Transplantation tolerance induced by intranasal administration of HY peptides Blood, May 15, 2004; 103(10): 3951 - 3959. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. M. Marelli-Berg, M. J. James, J. Dangerfield, J. Dyson, M. Millrain, D. Scott, E. Simpson, S. Nourshargh, and R. I. Lechler Cognate recognition of the endothelium induces HY-specific CD8+ T-lymphocyte transendothelial migration (diapedesis) in vivo Blood, April 15, 2004; 103(8): 3111 - 3116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Salio, M. J. Palmowski, A. Atzberger, I. F. Hermans, and V. Cerundolo CpG-matured Murine Plasmacytoid Dendritic Cells Are Capable of In Vivo Priming of Functional CD8 T Cell Responses to Endogenous but Not Exogenous Antigens J. Exp. Med., February 17, 2004; 199(4): 567 - 579. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. He, S. Schenk, Q. Zhang, A. Valujskikh, J. Bayer, R. L. Fairchild, and P. S. Heeger Effects of T Cell Frequency and Graft Size on Transplant Outcome in Mice J. Immunol., January 1, 2004; 172(1): 240 - 247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. James, J.-G. Chai, H. Dewchand, E. Macchiarulo, F. Dazzi, and E. Simpson Multiparity induces priming to male-specific minor histocompatibility antigen, HY, in mice and humans Blood, July 1, 2003; 102(1): 388 - 393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Yang and E. C. Lattime Tumor-induced Interleukin 10 Suppresses the Ability of Splenic Dendritic Cells to Stimulate CD4 and CD8 T-Cell Responses Cancer Res., May 1, 2003; 63(9): 2150 - 2157. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Krampera, S. Glennie, J. Dyson, D. Scott, R. Laylor, E. Simpson, and F. Dazzi Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide Blood, May 1, 2003; 101(9): 3722 - 3729. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Basta, W. Chen, J. R. Bennink, and J. W. Yewdell Inhibitory Effects of Cytomegalovirus Proteins US2 and US11 Point to Contributions from Direct Priming and Cross-Priming in Induction of Vaccinia Virus-Specific CD8+ T Cells J. Immunol., June 1, 2002; 168(11): 5403 - 5408. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and (Smcy, 
).
B and C, A total of 5 x
106 HY peptide (Db/Uty,
Db/Smcy, and Ab HY) coated
spleen cells were injected i.p., and B cell-depleted spleen cell
populations (14 days after immunization) were stained with anti-CD8
and Db/Uty tetramer and analyzed by FACS ex
vivo (B) and following in vitro restimulation
(C). D, ELISPOT analysis of
HY/Ab-reactive cells in B cell-depleted spleen cell
populations from responders in E and F.
Presence (+) or absence (-) of HY/Ab peptide on female APC
is indicated. Numbers of ELISPOT reactions per 105 cells
are plotted. E and F, A total of 5
x 106 
). B,
Specificity of HY-specific T cell clones isolated following
immunization with male DC (FP) or DC (FP) and skin graft.
C, WMHHNMLDI (Uty)-specific clones shown
in B stained differentially with tetramer; clone A10
reproducibly stained weakly with the cognate tetramer, whereas clone A4
was representative of the majority of clones.