Download Real-time T-cell profiling identifies H60 as a major

Plenary paper
Real-time T-cell profiling identifies H60 as a major minor histocompatibility
antigen in murine graft-versus-host disease
Eun Young Choi, Gregory J. Christianson, Yoshitaka Yoshimura, Nadja Jung, Thomas J. Sproule, Subramaniam Malarkannan,
Sebastian Joyce, and Derry C. Roopenian
Although CD8 T cells are thought to be a
principal effector population of graftversus-host disease (GVHD), their dynamics and specificity remain a mystery. Using a mouse model in which donor and
recipient were incompatible at many minor histocompatibility antigens (minor H
Ags), the CD8 T-cell response was tracked
temporally and spatially through the
course of GVHD. Donor CD8 T cells in the
circulation, spleen, lung, and liver demonstrated virtually identical kinetics: rapid
expansion and then decline prior to morbidity. Remarkably, up to one fourth of the
CD8 T cells were directed against a single
minor antigen, H60. Extreme H60 immunodominance occurred regardless of
sampling time, site, and genetic background. This study is the first to analyze
the T cells participating in GVHD in “real-
time,” demonstrates the exceptional degree to which immunodominance of H60
can occur, and suggests that such superdominant minor H Ags could be risk
factors for GVHD. (Blood. 2002;100:
4259-4265)
© 2002 by The American Society of Hematology
Introduction
Allogeneic bone marrow transplantation (BMT) after total body
irradiation is a common treatment for nonmalignant and malignant
hematologic disorders.1 However, it is greatly complicated by
graft-versus-host disease (GVHD), in which donor T cells generate
a response against host alloantigens. There are 2 critical gaps in
understanding the pathogenesis of GVHD. The first is in the
cellular progression of this disease. It is established that GVHD is a
consequence of donor CD4 and CD8 T cells causing damage to
target organs, such as skin, gut, liver, and lung.2,3 Alloreactive
donor CD8 T cells are thought to be responsible for much of the
tissue damage, but remarkably little is known concerning the
sequence of events by which this occurs.
The second gap is the antigenic basis of GVHD. In major
histocompatibility complex (MHC)–matched individuals, GVHD
is widely considered to be a consequence of T-cell responses
against minor histocompatibility antigens (minor H Ags).2,4 Given
that minor H Ags arise as a side product of naturally occurring
polymorphisms in a wide array of genes,5 hundreds to thousands of
them could be operative in any donor-recipient setting. A key issue,
therefore, is how many minor H Ags participate in the graft-versushost (GVH) response. Although CD8 T cells directed against an
immunodominant minor H Ag, HA-1, have been associated with
human GVHD,6 a thorough assessment of the immunodominant
minor H Ag issue would be best approached using mouse models.
Recent advances have led to the molecular identification of a
number of mouse minor H Ags that are immunodominant in certain
experimental settings7,8; however, there is a paucity of information
on mouse minor H Ags against which T cells are directed during
GVHD. Here, taking advantage of well-defined minor H Ag
immunogenetics and molecular information of minor H Ags, we
From the Jackson Laboratory, Bar Harbor, ME; Department of Microbiology
and Immunology, Vanderbilt University School of Medicine, Nashville, TN; and
Department of Medicine, Blood Research Institute, Milwaukee, WI.
Submitted May 2, 2002; accepted July 22, 2002. Prepublished online as Blood
First Edition Paper, August 22, 2002; DOI 10.1182/blood-2002-05-1299.
Supported by National Institutes of Health grants R01 HL56579, AI28802,
and HL54977.
BLOOD, 15 DECEMBER 2002 䡠 VOLUME 100, NUMBER 13
elucidate the specificity and dynamics of CD8 T cells involved in
systemic and local immune responses during GVHD and identify
H60 as a remarkably dominant minor H Ag in this disease.
Materials and methods
Mice
C57BL/6J (B6) female mice were used as donors of bone marrow (BM) and
spleen cells. C.B10-H2b/LiMcdJ (BALB.B), A.BY-H2bH2-T18b/SnJ (A.BY),
LP/J, 129P3/J 129 male mice were used as recipients. Congenic mouse
strains, B6.C-H60 c/Dcr,8 B10.CE-H13bAw(30NX)/Sn, B10.129-H46bH47b(21M)/
Sn, and B6.C-H28c/By, were used for establishment and maintenance of
cytotoxic T-lymphocyte (CTL) lines. Donor and recipient mice were 8 to 12
weeks old and were maintained under specific pathogen-free conditions.
Induction of GVHD
Recipient animals were irradiated with split doses of 450 cGy from a 137Cs
source with a 4-hour interval, and injected with BM and spleen cells from
B6 female mice 4 hours after the second irradiation. Irradiated BALB.B
mice not given BM typically died within 10 days. BM cells were flushed
from femurs and tibiae of the donor mice with phosphate-buffered saline
(PBS). A single-cell suspension of donor BM and spleen cells was filtered
through sterile nylon mesh, washed, and resuspended in PBS. Then,
5 ⫻ 106 BM cells and 2 ⫻ 107 splenocytes were admixed in PBS and
injected intravenously in a volume of 200 ␮L into a lateral tail vein.
Cell lines
Cell lines were maintained in Dulbecco modified Eagle medium (DMEM;
Life Technologies, Grand Island, NY) supplemented with 5% fetal bovine
Reprints: Derry C. Roopenian, Jackson Laboratory, 600 Main St, Bar Harbor,
ME 04609; e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology
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BLOOD, 15 DECEMBER 2002 䡠 VOLUME 100, NUMBER 13
CHOI et al
serum (FBS; Hyclone, Logan, UT). Transporter associated with antigen
presentation (TAP)–deficient human T2 cells expressing H-2Db (T2-Db)
and H-2Kb (T2-Kb) were provided by P. Cresswell (Yale University, New
Haven, CT). The H60b-specific H-2Kb–restricted CTL line SP/H60, the
H13b-specific H-2Db–restricted CTL line SP/H13, the H4b-specific H-2Kb–
restricted CTL clone M9,9 and the HY (Uty)–specific H-2Db–restricted
CTL clone CTL-1010 have been described. The H28-specific CTL line
B6.2/H28 was generated by immunizing C57BL/6J mice with cells from
H28 congenic B6.C-H28c/By mice. CTL lines were maintained by weekly
restimulation with appropriate stimulator cells and 50 U/mL recombinant
interleukin 2 (rIL-2).11
Cell-mediated lysis assay
A modified 51Cr release assay has been described.8 For peptide loading,
51Cr-labeled T2-Db and T2-Kb cells were incubated with a 100-nM
concentration of synthetic peptides for 30 minutes at 37°C and washed
twice with PBS. Synthetic peptides used were LTFNYRNL for H60,7
ILENFPRL for H28,12 SGIVYIHL for H4b (S.M., manuscript in preparation), SSVIGVWYL for H13b,13 and WMHHNMDLI for HY-UTY,14
respectively.
Purification of resident liver- and lung-infiltrating leukocytes
Isolation of liver and lung leukocytes was performed based on the
procedure of Masopust et al.15 To deplete circulating peripheral blood
leukocytes (PBLs), recipient mice were perfused with PBS/heparin (75
U/mL; Sigma, St Louis, MO) prior to being killed. Livers were pressed
through stainless steel mesh and suspended in 5% fetal calf serum
(FCS)–PBS. The cell suspensions were admixed with 33% Percoll containing 100 U/mL heparin and were then centrifuged 2000 rpm for 15 minutes
at room temperature. The pellets were resuspended in red blood cell (RBC)
lysis solution, washed twice, and resuspended in 5% FCS-PBS. Lungs were
cut into small pieces, washed with 1 ⫻ Hanks balanced salt solution
(HBSS)/HEPES (N-2-hydroxyethylpiperazine-N ⬘-2-ethanesulfonic acid; 10
mM), and were incubated in 20 mL digestion solution (1 ⫻ HBSS/HEPES,
150 U/mL collagenase, Sigma; 1 mM MgCl2, 1 mM CaCl2, and 5% FCS) at
37°C for 30 minutes. After washing twice with 5% FCS-PBS, the cells were
admixed with 33% Percoll and spun at 2000 rpm for 15 minutes. The cells
were washed and resuspended in 5% FCS-PBS.
Cell staining and flow cytometry
Fresh PBLs, splenocytes, or lymphocytes harvested from liver and lung
were incubated at 4°C for 50 minutes in staining buffer (1 ⫻ PBS with 0.1%
bovine serum albumin [BSA] and 0.1% sodium azide) containing phyco-
erythrin (PE)–labeled H60/H-2Kb, H13b/H-2Db, and HY/H-2Db tetramers
and saturating amounts of fluorescein isothiocyanate (FITC)–conjugated
anti-CD8 monoclonal antibody (mAb; 53-6.72, Jackson Laboratory) or
allophycocyanin (APC)–conjugated anti-CD8 (Pharmingen, San Diego
CA) and FITC-conjugated anti-CD11a (2D7; Pharmingen) mAbs. PElabeled tetramers for H60/H-2Kb (LTFNYRNL), H13b/H-2Db (SSVIGVWYC), and HY/H-2Db (WMHHNMDLI) were prepared as described.8 FITC-conjugated anti-␤2-microglobulin B (anti-␤2mb; S19.8,
Jackson Laboratory Flow Cytometry Facility) was used to distinguish
donor B6 (␤2-mb) from recipient (␤2-ma) cells.16 Other mAbs were
APC-conjugated anti–Mac-1 (M1/70; Pharmingen), Cy3-conjugated or
PE-conjugated anti-CD4 (GK1.5), FITC-conjugated anti–GR-1 (RB68C5), and PE-conjugated anti-CD8 53-6.72 (Jackson Laboratory Flow
Cytometry Facility). The stained cells were analyzed using a FACScan or
FACSCalibur equipped with CellQuest software (Becton Dickinson, San
Diego CA).
Results
Dramatic expansion of circulating and peripheral
organ-resident donor CD8 T cells during GVHD
To elicit acute minor H Ag–mediated GVHD, a B6 female donor
inoculum consisting of a mixture of BM and splenocytes (as a
source of mature T cells) was injected into MHC-matched (H-2b),
lethally irradiated BALB.B male recipient mice.17,18 Morbidity
started at day 21 after allogeneic transfer and reached about 75% by
60 days (Figure 1A). The allogeneic BALB.B recipients exhibited
typical clinical signs of acute GVHD (ruffled fur, diarrhea, and loss
of body weight), whereas B6 recipients of syngeneic B6 donor cells
remained healthy. To elucidate the temporal dynamics of the CD4
and CD8 T cells, PBLs were collected serially from recipients of
allogeneic or syngenic cells and analyzed for CD8/CD4 ratios.
Allogeneic donor leukocytes (distinguished from recipient cells
serologically by allelic expression of ␤2-m were rare in BALB.B
recipient blood up to about day 4 after adoptive transfer, suggesting
their sequestration in recipient tissues (Figure 1B). Donor CD4 and
CD8 T cells were readily detected by day 7 (Figure 1C; 100% of
CD8 T cells and 87.3% of CD4 T cells were donor-derived). The
CD8 T cells greatly outnumbered CD4 T cells between days 7 to 14
after transplantation and then decreased. In contrast, after syngenic
Figure 1. GVHD in B6 3 BALB.B mice. Splenocytes
(2 ⫻ 107) along with 5 ⫻ 106 BM cells from B6 female
mice were injected intravenously into irradiated (900
cGy) allogeneic BALB.B male (B6 3 BALB.B) or syngeneic (B6 3 B6) recipients. (A) Percent survival and mean
weight plot (number of survivors indicated for some data
points). (B) Recipient leukocytes disappear and transferred allogeneic donor leukocytes appear in the spleen
and liver before they are detected in the blood. B6
recipient cells were distinguished from donor cells using
the ␤2-mb allotypic marker. In 1B, squares indicate spleen
cells; diamonds, liver cells; and circles, PBLs. Open
symbols represent recipients and closed symbols represent donors. (C) Seven days after allogeneic transfer, all
CD8 cells and a majority of CD4 T cells in host blood are
of donor origin (␤2-mb⫹). (D) Kinetics of donor-derived
CD4 and CD8 T cells from host blood after allogeneic and
syngeneic transfer. Data are representative of 2 to 3
independent experiments.
BLOOD, 15 DECEMBER 2002 䡠 VOLUME 100, NUMBER 13
transfer, a normal CD8/CD4 ratio was maintained in both syngeneic BALB.B and B6 recipients (0.2 and 0.3-0.7, respectively;
Figure 1D).
Similar patterns were observed with resident leukocytes from
perfused lymphoid and nonlymphoid peripheral organs (Figures 1B
and 2). On day 3 after allogeneic transfer, spleen, liver, and lung of
perfused BALB.B recipients showed low cellularity, but ␤2-mb⫹
donor-derived CD4 and CD8 T cells were detected (Table 1). To
determine the order in which the donor CD4 and CD8 T cells
responded and proliferated, splenocytes and liver leukocytes were
harvested from perfused BALB.B recipients of CFSE (Scarboxyfluorescein diacetate succinimidyl ester)–labeled B6 splenocytes on day 4 after transfer. Donor CD4 T cells had undergone
more cell cycling, more than 3 doublings compared with CD8 T
cells (Figure 2A). Rapid expansion of B6 donor-derived resident
CD8 T cells in spleen, liver, and lung of BALB.B recipients then
occurred between days 3 and 7 (Table 1). The CD8/CD4 ratios of
liver and lung exactly paralleled those observed for spleen (Figure
2B), from 0.6 on day 7 to a peak of 3 on day 14 after transplantation
at which time CD8 T cells reached a peak of 37% to 40% (liver
and lung) and 8% (spleen) of leukocytes, and then declined (data
not shown).
The reduction of CD8 T cells was followed by a remarkable
increase in myeloid lineage cells. Although the myeloid cells
present immediately following transplantation were likely of both
donor and recipient origin (Figure 1B), they increased in frequency
to the extent that by day 28 after transplantation, 50% to 63% of
immune cells recovered from the liver and lung, respectively, were
Mac-1⫹, of which 90% were also Gr-1⫹ (Figure 2C,D), implying
an influx of mature granulocytes of donor origin into the organs of
allogeneic recipients. In contrast, BALB.B recipients of syngeneic
transfer showed no skewing toward Mac-1⫹ cells (not shown) and
maintained a normal CD8/CD4 ratio (0.3-0.5:1).
H60 IN GVHD
4261
Table 1. Leukocytes recovered from peripheral organs of GVHD mice*
Spleen
Liver
Lung
Day 3 after transplantation
2 ⫻ 106
3 ⫻ 105
1 ⫻ 105
% donor CD4 T cells†
11
4
15
% donor CD8 T cells†
35
52
56
Day 7 after transplantation
2.6 ⫻ 107
2.7 ⫻ 106
2.7 ⫻ 106
% donor CD4 T cells†
90
99
99
% donor CD8 T cells†
87
97
97
Day 14‡
6.4 ⫻ 107
6 ⫻ 106
4.3 ⫻ 106
Day 28‡
2.3 ⫻ 107
8.8 ⫻ 106
10.3 ⫻ 106
4.8 ⫻ 107
1.2 ⫻ 106
1.2 ⫻ 106
B6 female 3 BALB.B male
BALB.B male 3 BALB.B male
Day 14 after transplantation
*Means of nucleated cells from 3 mice.
†Percent of donor-derived T cells in total CD4 or CD8 T cells. Donor B6 cells were
distinguished from recipient BALB.B cells by allelic expression of ␤2-m: B6, ␤2-mb;
BALB.B, ␤2-ma.
‡About 100% of lymphocytes were donor-derived. Data are representative of 2
experiments.
Dominance of H60-specific CD8 T cells during GVHD
To determine the specificities of circulating CD8 T cells during
GVHD, serial PBL samples pooled from 10 allogeneic BALB.B
recipients were analyzed by flow cytometric analysis using PEconjugated tetramers for H60/H2-Kb, H13b/H2-Db, and HY/H2-Db
minor H Ag/MHC complexes along with FITC-conjugated antiCD8 mAb. The frequency of H60 tetramer-positive CD8 T cells
increased dramatically from undetectable on day 4 to 9% of CD8 T
cells on day 10, then declined to 1% to 2% of CD8 T cells on day 30
after transplantation (Figure 3A). (Because GVHD mortality began
on day 21, the results after that day were biased in that they were
from PBLs of survivors.) In contrast to readily detectable H60specific cells, only a small percentage of H13 tetramer–binding
Figure 2. Kinetics of donor-derived resident CD4 and CD8 T cells from lymphoid and nonlymphoid target organs of perfused GVHD mice. (A) Analysis of spleens and
livers 4 days after allogeneic transfer of CFSE-labeled B6 female donor splenocytes plus unlabeled BM into BALB.B male recipients. (B) CD8/CD4 ratios of lymphocytes from
spleens, livers, and lungs of hosts after allogeneic (B6 3 BALB.B) and syngeneic (BALB.B 3 BALB.B) transfer. (C) Resident lung leukocytes from B6 3 BALB.B mice on day
7 and day 28 after transplantation were analyzed by forward scatter (FSC) and side scatter (SSC) after staining with anti–Mac-1 and –Gr-1 antibodies. Most of the large
granular cells on day 28 were Mac-1⫹ and Gr-1⫹. (D) Percentages of Mac-1⫹/Gr-1⫹ lung, liver and splenic leukocytes were determined at time points after allogeneic transfer.
In panels A to D, data are from pooled samples from 3 mice and are representative of 3 independent experiments.
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CHOI et al
BLOOD, 15 DECEMBER 2002 䡠 VOLUME 100, NUMBER 13
Figure 3. H60 dominates the CD8 T-cell response
regardless of timing and tissue site. (A) Serially
obtained PBLs from male BALB.B recipients of allogeneic female B6 cells were stained with anti-CD8 mAb and
tetramers for H60, H13, and HY minor H Ags and
analyzed by flow cytometry. Data are from pooled samples
from 10 mice and are representative of multiple experiments. (B) Lymphocytes from the spleens, livers, and
lungs from groups of 3 perfused male BALB.B recipients
of female B6 cells were prepared at each time point after
transfer, stained with the indicated reagents, and analyzed by flow cytometric analysis. Data are representative of 2 independent experiments.
cells was detectable on days 10 and 14 after transplantation and no
HY tetramer–positive CD8 cells were detected.
To determine the specificities of the CD8 T cells residing in
GVH target organs, resident leukocytes from perfused liver, lung,
and spleen were stained with anti-CD8 mAb-APC–, anti-CD11a
mAb-FITC–, and PE-conjugated tetramers for H60, H13, and HY
minor H Ags (Figure 3B). H60-tetramer–binding CD8 T cells
expanded between days 3 and 7, peaking at 18% to 23% of CD8 T
cells on day 7, followed by an approximate 8-fold drop to 3% to 4%
of CD8 T cells by day 21 after transplantation. All H60 tetramer–
positive cells showed a CD11ahigh phenotype, indicating prior
activation. In contrast, a low frequency of H13 tetramer–positive
CD8 T cells was detected only from spleens (4%), whereas no HY
tetramer–positive cells were detected. These combined profiling
experiments demonstrated that in the B6 3 BALB.B GVHD
model the immune response to H60 dominated temporally and
spatially over responses to H13 and HY. Additionally, the approximate doubling of the frequency of H60 tetramer–positive CD8 T
cells in the peripheral organs versus PBLs suggests that H60
immunodominance was intensified at these sites.
To assess the functional activity of minor H Ag–specific CD8 T
cells during development of GVHD, specific cytolytic potential
was evaluated. Ex vivo splenocytes harvested on day 10 after
allogenic transfer were predominantly blast cells. Consistent with
the apoptosis and anergy usually associated with excessive antigen
stimulation,19-21 these cells yielded low but significant cytolytic
activity against H60 peptide–loaded T2-Kb target cells (12%
specific lysis) without any specific cytotoxicity against other minor
H Ags (data not shown). To enrich CD8 T effector cells, splenic
leukocytes harvested 7 and 10 days after allogeneic transplantation
were cultured in vitro with irradiated BALB.B male splenocytes in
the presence of exogenous IL-2. The effector cells were then tested
for cytolytic activity against T2-Kb or T2-Db target cells loaded
with synthetic peptides corresponding to a number of known minor
H epitopes, including those known to act as dominant minor H Ags
(H28 and H4). Only H60-specific cytolytic activity was detected
(Figure 4A). Similar results were obtained from the analysis using
lymphocytes from spleen, liver, and lung of perfused BALB.B
recipients. In all cases, appreciable H60-specific cytotoxic activity
was detected, with the highest levels (55%) from lung leukocytes,
whereas no significant cytotoxicity was detected against H28, H13,
H4, or HY peptide–loaded targets (Figure 4B). These results
suggest that H60 dominates during the GVH response to the extent
that it precludes the generation of CTLs against minor H Ags,
which themselves are known to behave in a dominant fashion in
other experimental settings.8,12,22
The H60-specific response dominates in GVHD regardless
of recipient-donor combination
A critical issue concerning immunodominant minor H Ags in
GVHD is whether H60 dominates in diverse donor-recipient
combinations. To test this possibility, BM and splenocytes from B6
female mice were transferred into H-2b–matched mouse strains of
diverse genetic origins: A.BY, LP/J, and 129, along with BALB.B
mice. PBLs from 10 recipient cohorts were pooled and tested for
tetramer staining. All the recipients showed a frequency of H60
tetramer–positive CD8 T cells indistinguishable from BALB.B
recipients (Figure 5). One peculiarity was that even though HY has
been known as an easily dominated antigen,23,24 HY-specific
responses dominated over the responses to H13 in A.BY and 129
recipients with a little deviation from typical immune response
kinetics. This result suggests that variation in background genetics
can alter the immunodominance hierarchy of subordinate minor H
Ags; however, the dominance of the H60-specific immune response was invariant.
Discussion
Temporal and spatial profiles of the GVH response
after allogeneic BMT
Although it is well established that CD4 and CD8 T cells contribute
to the GVHD process, remarkably little is known concerning the
BLOOD, 15 DECEMBER 2002 䡠 VOLUME 100, NUMBER 13
H60 IN GVHD
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However, it remains to be established how this pathology comes
about. Recipient macrophages have been shown to be important for
promoting the early stages of GVHD.27 In addition to cell-mediated
cytotoxicity (perforin- and Fas-mediated),28 cytokines (ie, tumor
necrosis factor ␣, interferon ␥, and IL-12) released by the highly
activated effector CD8 T cells could also contribute to acute GVHD
as direct soluble mediators or by recruiting other cells such as
macrophages and granulocytes to the GVHD sites.29 The decline in
CD8 T cells observed after their peak is consistent with clonal
exhaustion as a consequence of activation-induced cell death.30 The
progressive accumulation of donor granulocytes and other myeloid
lineage cells in target organs may exacerbate that damage, leading
to morbidity.31
Immune responses are generally thought to occur in the
peripheral lymphoid organs, followed by release into the circulation. It is thus surprising that the CD8 T-cell kinetics in nonlymphoid target organ, including H60-specific T cells, paralleled or
even preceded those found in the circulation. It is possible that at
least some CD8 T cells used GVHD target organs as sites for
ectopic activation and clonal expansion, leading to immediate
organ damage. Alternatively, the immediate accumulation of
activated CD8 T cells in target organs may reflect very efficient
lymph organ egress and recruitment mediated by GVHD- and
Th1-associated chemokines and their receptors.32,33
H60 is a dominant minor H Ag in mouse GVHD
Figure 4. Cell-mediated lympholysis (CML) analysis of cells from GVHD mice.
(A) Analysis of spleens harvested from BALB.B recipients of B6 allogeneic cells on
days 7 and 10 after transfer. Splenocytes were cultured in mixed leukocyte culture
(MLC) with irradiated splenocytes from male BALB.B stimulator cells. The effector
cells were tested for their ability to lyse H60, H28, H4, H13, and HY minor H
Ag-peptide loaded, 51Cr-labeled T2-Kb or T2-Db target cells. Minor H Ag–specific CTL
lines, SP/H60, B6.2/H28, SP/H13, M9, and CTL-10 CTL lines were included as
positive controls. (B) Analysis of spleens, livers, and lungs harvested from perfused
allogeneic BALB.B recipients on day 10 after transplantation. Data are from pooled
organs from 3 mice. Minor H Ag–specific CTL lines were included as positive controls.
cellular progression involved. Our T-cell profiling studies clarify
this issue by suggesting a predictable sequence of events that
precede the morbidity associated with acute GVHD. Soon after
transplantation, donor T cells were immediately sequestered from
the circulation (Figure 1B). This was a direct consequence of
alloantigenic stimuli because syngeneic cell transfer yielded circulating T cells immediately (data not shown), and therefore, this is
likely to have been a consequence of T-cell activation–dependent
up-regulation of adhesion molecules such as CD44 and leukocyte
function-associated antigen 1 (LFA-1).25 Donor CD4 T cells were
the first to undergo appreciable clonal proliferation (Figure 2A); in
doing so, they presumably provided help to facilitate subsequent
CD8 T-cell proliferation. Donor CD8 T cells then proliferated and
rapidly outnumbered CD4 T cells resulting in a CD8/CD4 T-cell
ratio inversion. This temporal relationship is consistent with an
established requirement for both CD4 and CD8 T cells in acute
GVHD across minor H Ag disparities,26 and is likely a requirement
to amplify the low precursor frequency of naı̈ve minor H Agspecific CD8 T cells to numbers sufficient to cause pathology.
Although there have been efforts to elucidate the minor H Ags that
participate in GVHD in the mouse model,17,22 the preponderance of
information regarding the specificity of CD8 T cells involved in the
GVH response is derived from human studies in which CD8 T cells
were isolated from patients with GVHD.6,34 One of those, HA-1,
has been implicated in a risk factor for severe GVHD in Dutch and
US populations,35 but not in a Japanese population.36 A key
unsolved issue is whether, among a large number of potential minor
Figure 5. H60 immunodominance occurs on different genetic backgrounds.
H-2b–matched A.BY, BALB.B. LP/J and 129/J male mice were injected with a mixture
of splenocytes and BM cells from female B6 mice. After allogeneic transfer, pooled
PBLs from 10 recipients were collected serially and stained with anti-CD8 mAb and
tetramers for H60, H13, and HY minor H Ags. Data are representative of 3
experiments.
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CHOI et al
H Ag disparities that distinguish a typical MHC-matched donor and
recipient, there are immunodominant antigens that could account
for a disproportionate percentage of the GVH response.
This study is the first to identify a mouse minor H Ag that is a
natural target of the GVH CD8 T-cell response. Importantly, H60
immunodominance was manifested on genetically varied backgrounds with diverse sets of subordinate minor H Ags.37 BALB.B
mice differ from male B6 at an array of molecularly defined minor
H Ags, including H13, H4, H7, H28, H60, and HY. Immunization
of normal B6 mice with BALB.B cells results in the generation of
CD8 T cells directed primarily at the immunodominant H60, H28,
H4, and H7 minor H Ags.8 Our results are striking in the degree to
which H60 immunodominance is exaggerated in the GVHD model
at the expense of other minor H Ags, including those such as H28
and H4 that exhibit the properties of immunodominance in other
experimental settings. We have additionally observed that H7,
which has been implicated in GVHD,22 is similarly subordinate to
H60 (data not shown). Although we have shown that a surprisingly
high percentage of the CD8 T-cell response is directed against the
H60 minor H epitope, it remains to be determined whether H60
poses a survival risk for GVHD.
The mechanism by which H60 achieves its extreme immunodominance in the GVH setting is a matter of speculation. It is
intriguing to consider the possibility that this attribute is related to
the natural in vivo function of the H60 glycoprotein as a ligand for
the stimulatory natural killer (NK) receptor (NKG2D), engagement
of which could provide activation/costimulatory signals to NK
cells and CD8 T cells and consequent enhancement of immunity.38
Interestingly, the H60 minor H octamer peptide partially inhibits
NKG2D from binding the H60 glycoprotein, suggesting that the
peptide encompasses a critical NKG2D-binding site.39 However, it
is difficult to envision how the H60 protein could lead to
dominance specific for the H60 minor H peptide-driven CD8 T-cell
immune response. It is more likely that H60 immunodominance is
a property of T cells responding in a more conventional manner
to the H60 peptide/Kb complex.40
In summary, our study shows the extreme level to which
immunodominance can assert itself in a GVH setting and supports
the concept that a single dominant minor H Ag can play an
unexpectedly important role in the GVH response.
Acknowledgments
We thank Leo Lefrancios for sharing his liver and lung leukocyte protocols and Elizabeth Simpson and David Serreze for
editorial comments.
References
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Erratum
In the article by Underhill et al entitled “IgG plasma cells display a unique
spectrum of leukocyte adhesion and homing molecules,” which appeared in
the April 15, 2002, issue of Blood (Volume 99:2905-2912), the name of the
second author should appear Heather A. Minges Wols.