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Kimmel Cancer Institute, Jefferson Medical College, Philadelphia, PA 19107
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB.B
TDL did not indicate expansion of any particular TCR Vß family,
whereas Vß10 and Vß14 families were significantly expanded in the
B6->CXBE TDL. However, PCR-based complementarity-determining region 3
size spectratyping revealed overlapping involvement of donor Vß1, 6,
8, 9, 10, and 14 families in both BALB.B and CXBE recipients and unique
utilization of the Vß4 family in BALB.B mice, suggesting oligoclonal
T cell responses to a limited number of miHA. In addition, the
injection of CD8+Vß14+ B6 T cells into
irradiated BALB.B and CXBE mice induced lethal GVHD, confirming the
involvement of miHA-specific T cells within an individual Vß family. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Multiple minor histocompatibility Ags (miHA) play an important role in
the induction of GVHD following MHC-matched allogeneic BMT (8, 9, 10, 11).
MiHA are presented by MHC class I and class II molecules (9, 12), the
former of which are recognized by CTL that can be generated in vitro by
secondary stimulation of primed effector T cells (13). As an
experimental system, the T cell response of C57BL/6By (B6) mice
directed against miHA expressed by H2b-matched BALB.B mice
has been thoroughly investigated. Despite estimated differences of more
than 29 miHA loci between these two strains, it was found that the in
vitro CTL response was directed to only a few immunodominant Ags (14).
These BALB.B miHA were detected because of their differential
expression in a panel of target cells from the CXBE, G, I, J and K
recombinant inbred (RI) strain, generated from F1 crosses
from an original B6 and BALB/c mating. The hierarchy of the in vitro B6
CTL reactivity indicated that CXBG and CXBK strains expressed
first-order immunodominant miHA, whereas CXBE expressed second-order
miHA which could stimulate a response only in the absence of the
first-order Ags (14). However, GVHD studies involving the
transplantation of B6 T cells and marrow into irradiated BALB.B and CXB
RI strains indicated that the in vitro immunodominant miHA hierarchy
did not correlate with GVHD potential (15). In contrast to strong GVHD
responses observed in the BALB.B and CXBE strains, GVHD was not evident
in the CXBG and CXBK recipients. An interstrain GVHD response analysis
with the BALB.B and CXB RI strains suggested that a minimum of two
distinct MHC class I-restricted miHA (or groups of miHA) could account
for induction of disease in the parental B6BALB.B combination,
designated GVH-1 and GVH-2 (16). Consistent with the observed
importance of MHC class I-restricted miHA, all positive B6 GVHD
responses in the BALB.B and CXB RI strains were found to involve
mediation by CD8+ T cells, most of which appeared to be
dependent on CD4+ T cell help for their generation; in
BALB.B mice, CD4+ T cells also independently caused a high
level of lethal GVHD (17). Furthermore, a recent phenotypic study of
the B6 CD4+ TCR Vß repertoire during the early
development of GVHD in BALB.B and CXBE mice has given the first
indication of involvement of a limited anti-host miHA response
(18).
In the current investigation, we approached the more critical question
of the nature and extent of the B6 CD8+ T cell GVHD
response to miHA in the BALB.B and CXBE recipients. Positively selected
miHA-specific T cell blasts were collected from the thoracic duct
lymphocyte (TDL) pool of irradiated BALB.B and CXBE mice, 5 days after
transplantation of host-primed B6 T cells. Initial flow cytometric
phenotype analysis of the CD8+ T cells in the B6BALB.B
TDL did not suggest significant expansions in any TCR Vß family,
whereas B6 CXBE TDL displayed significant expansions of the Vß10
and Vß14 families.
TCR Vß repertoire complexity was further examined by the highly
sensitive PCR-based complementarity-determining region 3 (CDR3) size
spectratyping analysis, in which the bulk TCR sizes for any given Vß
family of a control population exhibit a Gaussian distribution ladder
of in-frame expressible bands separated by three bases (19, 20). A
skewing of the normal size distribution, reflected by increased band
intensity, is indicative of an expanded CDR3 size expression and
suggests an oligoclonal T cell response (21). Characterization of the
TCR Vß repertoire of the responding donor CD8+ T cells is
the first step toward ultimate identification of the specific T cells
involved in the GVHD response. By this approach, we have found that
B6BALB.B TDL T cells exhibited biased CDR3 size usage in the Vß1,
4, 6, 8, 9, 10, and 14 families, whereas B6 CXBE TDL T cells
exhibited overlapping usage of the Vß1, 6, 8, 9, 10, and 14 families.
The Vß4 response appeared to be unique in recognition of miHA
expressed only by BALB.B recipients. The implications of these findings
are that GVHD in this model system seems to involve an oligoclonal
response to a limited number of immunodominant miHA. Furthermore, these
miHA appear capable of inducing T cell responses in both the CXBE RI
and BALB.B parental strains, with no evidence of competitive inhibition
as previously observed in vitro (14). In addition,
CD8+Vß14+ B6 T cells displayed high GVHD
potential upon transplantation into irradiated BALB.B and CXBE mice,
supporting the involvement of T cells from an individual Vß family in
the development of GVHD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C.B10-H2b/LiMcdJ (BALB.B), CXB-2/By (CXBE), and C57BL/6By (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and/or raised in our breeding colony from breeder pairs provided by The Jackson Laboratory. For all experiments, sex-matched mice were used as donors and recipients between the ages of 7 and 16 wk. Mice were kept in a pathogen-free environment in autoclaved microisolator cages and were provided with acidified (pH 2.5) water and autoclaved food ad libitum.
Media
PBS (BioWhittaker, Walkerville, MD) supplemented with 0.1% BSA (Sigma Chemical, St. Louis, MO) was used for all in vitro manipulations of the donor bone marrow cells and lymphocytes. RPMI 1640 (Mediatech, Herndon, VA) supplemented with 10% FCS (Sigma), 2 mM L-glutamine (Mediatech), 50 IU/ml penicillin (Mediatech), 50 µg of streptomycin (Mediatech), and 5 IU/ml heparin sulfate (Schein Pharmaceutical, Florham Park, NJ) was used for TDL collection. PBS supplemented with 1% BSA and 0.1% NaN3 was used as a medium for staining cells for flow cytometry (FACS media). PBS alone and supplemented with 1% BSA was used during magnetic cell sorting.
Irradiation
Recipient mice received a lethal dose of whole body irradiation (825 cGy) from a Gammacell (Atomic Energy of Canada, Kanata, Ontario, Canada) 137Ce source at a dose rate of 116 cGy/min.
Monoclonal antibodies
Ascites fluid for anti-Thy-1.2 (J1j, rat IgM (22), anti-CD4 (RL172, rat IgM (23), and GK1.5, rat IgG (24)) mAb were used for cell preparations. Affinity-purified goat anti-mouse IgG (whole molecule) Ab was purchased from Cappel-Organon Teknika (Westchester, PA). For magnetic cell sorting and flow cytometric analyses, all mAb were purchased from PharMingen (San Diego, CA) and included FITC-conjugated anti-CD4 (clone RM4-5; no. 01064D) and anti-CD8 (clone 53-6.7; no. 01044D) mAb and biotinylated mAb specific for Vß2, 3, 4, 5.1/5.2, 6, 7, 8.1/8.2, 9, 10, 11, 12, 13, and 14.
Preparation of donor cells
Bone marrow cells were flushed from the femurs and tibias of B6 donor mice with PBS + 0.1% BSA and washed. To prepare anti-T cell-depleted bone marrow (ATBM), cells were incubated with J1j mAb (1:100 dilution of ascites fluid) and guinea pig C (1:30 dilution) in 6 ml of PBS + 0.1% BSA at 37°C for 50 min, and washed three times. ATBM was adjusted to 1.6 x 107 cells/ml in PBS for i.v. injection (0.1 ml) into recipients. T cell-enriched donor cells were prepared from pooled spleen and lymph node cell suspensions from appropriate BALB.B or CXBE primed and boosted B6 mice (i.p. injection of 1.5 x 107 spleen cells 2.5 wk apart). The cells were washed and resuspended in Gey’s balanced salt solution containing 0.7% NH4Cl to remove RBC. The cells were washed twice and filtered through a cell strainer to remove dead cells. B cells were removed by panning the cell suspension on goat anti-mouse IgG precoated plastic petri dishes for 1 h at 4°C, as previously described (16). The nonadherent (whole T cell enriched) cells were harvested. Cell purity was >85% positive for CD3 expression, as determined by flow cytometry. For induction of GVHD, whole T cells were depleted of CD4+ T cells by incubation with RL172 mAb (1:100 dilution of ascites fluid) and guinea pig C (1:30 dilution) in 6 ml of PBS + 0.1% BSA at 37°C for 50 min and washed three times. CD8+ enriched cells were incubated with either biotinylated or FITC-conjugated mAb specific for Vß14 in PBS for 30 min at 4°C. Cells were washed twice in PBS and incubated with MACS Magnetic streptavidin microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) for 30 min at 4°C. The cells were washed twice, resuspended in PBS + 0.1% BSA, and positively selected by either fluorescent cell sorting using an EPICS ELITE flow cytometer (Coulter Electronics, Hialeah, FL) or the Vario MACS Column System (Miltenyi). CD8+Vß14+ T cells were washed twice and resuspended in PBS for i.v. injection. For the GVHD experiments with CXBE recipients, CD4+ T cells were not depleted from the donor cell populations.
GVHD assay
BALB.B or CXBE mice were lethally irradiated (825 cGy) and 
6
h later injected i.v. (in a volume of 0.5 ml of PBS) with the
appropriate preparation of ATBM (2 x 106) alone
or in conjunction with either TDL or the experimental cell preparation
to be tested. Mice were checked daily for morbidity and mortality and
weighed twice per week until the experiments were terminated.
Statistical comparisons between groups were performed by the Wilcoxon
nonparametric test.
Thoracic duct cannulation
Thoracic duct cannulation was used to obtain positively selected host miHA-specific T cells, as previously described (18, 25). BALB.B or CXBE mice (3–4 mice/group) were lethally irradiated (825 cGy) and injected i.v. 6 h later with unseparated T cells (1–1.5 x 107 cells in 0.25 ml of PBS) from host-primed donor B6 mice and were cannulated 5 days later. Briefly, mice were anesthetized by an i.p. injection of avertin (1:50 dilution in PBS of a stock solution of 10 g of tribromoethanol in 10 ml of tert-amyl alcohol; Aldrich Chemical, Milwaukee, WI), an i.v. saline line inserted in the tail, and a cannula (PE-50 intramedic tubing; Clay Adams, Parsipanny, NJ) surgically implanted into the thoracic duct. TDL were collected over a 20-h period and the cells pooled from individual mice of each group.
Flow cytometric analysis of TCR Vß expression
For fluorescent staining, cells were washed, resuspended in FACS
medium, and incubated for 5 min at 4°C with culture supernatant
containing anti-FcR
ll mAb (clone 2.4G2, HB197; American Type
Culture Collection, Rockville, MD; (26)) to prevent nonspecific
staining by mAb. An isotype-matched FITC-conjugated mAb was used as a
negative control. TDL T cells were assayed for the percentage of Vß
expressing CD8+ T cells by two-color flow cytometry.
Cells were first incubated with each biotinylated anti-Vß mAb in
combination with FITC-anti-CD8 mAb for 30 min at 4°C. The cells
were then washed twice and incubated with PE-streptavidin (Caltag, San
Francisco, CA) for 30 min at 4°C, washed twice, and fixed in 1%
paraformaldehyde. Cells were analyzed on a EPICS C flow cytometer
(Coulter Electronics). Data from individual replicate experiments were
pooled, and statistical significance was determined by the Mann-Whitney
Wilcoxon rank test.
Preparation of RNA and cDNA
CD8+ T cells were first prepared by depletion
of CD4+ T cells via panning of TDL cell suspensions on
GK1.5 mAb (1:20 dilution of ascites fluid in PBS + 0.1% BSA)
precoated plastic petri dishes for 1 h at 4°C. This procedure
yielded a restricted population with 
92% CD8+ cells.
Total cellular RNA was then generated from 106 to
107 nonadherent CD8+ cells by homogenization in
1 ml of Ultraspec (Biotecx Laboratories, Houston, TX), separating
cellular DNA and protein by the addition of a 1:5 volume of chloroform,
vortexing for 5 s, and centrifuging at 12,500 rpm for 15 min. The
aqueous phase was transferred to a clean Eppendorf tube, and RNA was
precipitated at 4°C by adding an equal volume of isopropanol and
centrifuging at 12,500 rpm for 15 min. The supernatant was removed, and
the pellet was washed with 75% ethanol in diethyl pyrocarbonate (DEPC)
treated water and centrifuged, as above. The RNA pellet was resuspended
in 25 µl of DEPC water, heated to 55–65°C for 10 min and stored at
-20°C. Recovery of RNA was determined by spectrophotometry. The
poly(A)+ portion of the total RNA was converted into cDNA
using oligo(dT) as a primer for reverse transcription. Two micrograms
of total RNA in a volume of 9.5 µl were heated to 70–80°C,
centrifuged briefly, and placed on ice. Master mix (17.5 µl),
containing 1 µl of RNasin (40 U/µl), 6 µl of 5x Maloney murine
leukemia virus reverse transcriptase reaction buffer, 3 µl of
oligo(dT) primer (20 mM), 1.5 µl of deoxynucleotide triphosphates A,
G, C, T (25 mM each), and 3 µl of Maloney murine leukemia virus
reverse transcriptase (300 U/µl), was incubated at 37°C for
1.5 h to synthesize cDNA. All reagents were purchased from Promega
(Madison, WI). The reaction mixture was then heated to 95°C for 3
min, and the cDNA was stored at -20°C. PCR was performed using
murine ß-actin primers to establish the quality of the cDNA.
PCR amplification of cDNA and CDR3 size spectratyping
PCR was performed using a labeled constant primer (Cßb) and a
Vß primer specific for each Vß family to be analyzed. Cßb was
labeled using polynucleotide kinase (Promega) and
[-32P]ATP (Dupont-NEN, Boston, MA). All the
primers used have been previously described (27) with the exception of
the Vß20 primer, which was designed with a sequence of
GGTCAAGGAGAGATTCTCAGCTGT. Five microliters of 10x Taq
polymerase buffer B (Promega) were added to 5 µl of cDNA plus 2 µl
of MgCl2 (25 mM), 4 µl deoxynucleotide triphosphates A,G,
C, T (25 mM each), 2.5 µl of Vß sense primer (20 µM), 4 µl
Cßb labeled antisense primer (12 µM), 28 µl DEPC-treated water,
and 1 µl of Taq polymerase (2–5 U/µl, Promega). Thirty
cycles of amplification were conducted and on completion of PCR, 50
µl of 2x loading buffer containing 95% formamide, 20 mM EDTA,
0.05% bromphenol blue,, and 0.05% Xylene Cyanol FF was added to each
reaction. PCR reactions were either stored at -20°C or
electrophoresed. Each reaction tube was heated to 70°C for 3 min, and
6 µl of each were applied to a prewarmed 6% acrylamide sequencing
gel, as previously described (20). The gels were run at 55 mA for
1.5 h to maximize band resolution. Sequencing gels were dried, and
autoradiography was generally performed for 15 h at room
temperature without intensifying screens. Densitometric scanning of
autoradiographs was performed on a Personal Densitometer SI using
ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The
interpretations of the spectratypes were based on the criteria
previously established by Gorski et al. (19, 20). The relative band
intensities within a given spectratype were examined and compared with
the intensity patterns in the spectratype of the naive B6 control
group. The other bands within a given spectratype act as the internal
controls for any variability due to the PCR expansion or to different
sample sizes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B6 T cells (1–1.5 x 107) were injected into irradiated (825 cGy) BALB.B and CXBE mice for induction of GVHD. It was necessary to use cells from donor mice that had been presensitized (primed and boosted by i.p. injection of 1.5 x 107 spleen cells) to the appropriate recipient type to generate any significant TCR Vß family expansion of the CD8+ T cell population within the first week posttransplantation. The recipients underwent thoracic duct cannulation on day 5, and TDL was collected over a 20-h period and pooled from three to four mice per group. The positively selected TDL were >95% CD3+ and consisted of 55 to 67% CD4+ and 33 to 45% CD8+ T cells. A significant percentage (20–25%) of the TDL were blast-like in size, in contrast to TDL collected from B6 mice transplanted with T cells (1–1.5 x 107) from syngeneic-presensitized B6 mice, which yielded few blast-like cells and were >98% CD4+. Therefore, the TCR Vß repertoire analyses of TDL CD8+ T cells from GVHD recipients were compared with the TCR Vß repertoire of normal B6 splenic CD8+ T cells.
TCR Vß repertoire analysis by flow cytometry
TCR Vß repertoire analysis was conducted by two-color flow
cytometry of the TDL using a panel of anti-Vß mAb along with
anti-CD8 mAb. Of the 13 Vß families for which specific mAb were
available, positively selected B6BALB.B TDL did not display any
significant Vß family expansion (Fig. 1
). On the other hand, the B6CXBE TDL
exhibited significant expansions of the Vß10 (6.1% to 9.9%;
p < 0.05) and Vß14 (3.3% to 6.8%;
p < 0.05) families; and marginal expansion of the
Vß11 family (8.7% to 11.8%; p > 0.05), relative to
naive B6 splenic CD8+ T cells (Fig. 1).
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BALB.B TDL T cells
B6BALB.B TDL T cells (1.4 x 107) were
transplanted along with 2 x 106 ATBM into lethally
irradiated (825 cGy) BALB.B or CXBE recipients to demonstrate that
these cells were capable of inducing GVHD. Control recipients received
an injection of 2 x 106 ATBM alone. By day 25, both
the BALB.B and CXBE recipients exhibited the clinical symptoms of GVHD,
including diarrhea, ruffled fur, and weight loss (>20%), compared
with control ATBM mice (Fig. 2A). All of the BALB.B
recipients of TDL succumbed by day 50 (MST of 30 days), more rapidly
than the CXBE recipients which died by day 72 (MST of 50 days; Fig. 2B).
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The CDR3 sequence encoding the variable ß-chain V-D-J junctional
region defines the unique TCR clonotype(s) specific for a miHA and acts
as a fingerprint for the T cell lineage(s) bearing it. PCR-based
analysis, known as CDR3 size spectratyping (19, 20), was used to
determine the nature of the Vß family repertoires observed in the
B6BALB.B and B6CXBE CD8+ TDL, as a reflection of
the initial GVHD response. The Vß bands of a spectratype from normal
B6 splenic CD8+ T cells followed a Gaussian distribution
(Fig. 3A),
with the center bands of median CDR3 length being most intense and the
outer bands of longer and shorter lengths being less intense. In
contrast, the Vß band patterns of skewed spectratypes from TDL did
not exhibit Gaussian distributions, with some bands being more intense
relative to the same band distribution in the B6 control sample
(exemplified in Fig. 3). The CDR3 size spectratypes of 17 Vß families
expressed in B6 mice (Vß1-16 and Vß20) were examined for band
skewing suggestive of an oligoclonal expansion of particular T cell
lineages. The results (summarized in Table I) supported the findings of the
phenotypic analysis; i.e., Vß6 exhibited biased CDR3 usage in the
B6 BALB.B CD8+ TDL and Vß6, Vß10, and Vß14 were
skewed in the B6CXBE CD8+ TDL (Fig. 3). In addition, the
spectratype analysis indicated that both strain combinations exhibited
skewing of bands in the Vß1, 6, 8, 9, 10, and 14 families. In all
cases, the same CDR3 size band was enhanced in the same Vß family
between the two groups (Fig. 3). However, in the Vß14 family,
additional bands were enhanced in the B6 BALB.B CD8+ TDL
spectratype which were not skewed in the B6CXBE CD8+.
Most notably, biased CDR3 usage in the Vß4 family was unique to the
B6BALB.B CD8+ TDL, suggesting a response to a
BALB.B-specific miHA.
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Fluorescent cell sorting was used to positively select
CD8+Vß14+ T cells from
CXBE-presensitized B6 mice which were then transplanted into irradiated
(825 cGy) BALB.B recipients to demonstrate that cells from a single
Vß family were capable of inducing GVHD. Recipients were injected
with either the combination of 3 x 106 naive B6
CD4+ T cells (to provide Th function) and 2 x
106 ATBM alone or with either 4 x 106
CXBE-presensitized unseparated B6 CD8+T cells or 1 x
105 CD8+Vß14+-enriched T cells
(>99% Vß14+). Recipients of either the unseparated or
Vß14+CD8+ T cells exhibited the clinical
symptoms of GVHD between days 45 and 50, including weight loss (22 and
30%, respectively; Fig. 4A). By day 76, 60%
(MST of 49 days) of the BALB.B mice that received the unseparated
CD8+ T cells and 80% (MST of 67 days) of those given the
Vß14+CD8+ T cells had died, compared with
20% mortality in the ATBM control group (Fig. 4B).
In a similar manner, lethally irradiated (825 cGy) CXBE mice were
injected with 2 x 106 ATBM either alone or with
either 3 x 107 CXBE-presensitized unseparated or
3.5 x 106 Vß14+ enriched (>84%
Vß14+ selected by magnetic cell sorting) B6 T cells. Both
groups of CXBE recipients that received T cells exhibited the clinical
symptoms of GVHD by day 40, including weight loss (17 and 25%,
respectively; Fig. 5A).
By day 45, mice that received injections of
Vß14+-enriched CD8+ T cells had 80% fatality
(MST of 48 days), and by day 70, the mice that had been injected with
unseparated CD8+ T cells had 40% fatality, as compared
with 100% survival in the control ATBM group (Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB.B and B6CXBE transplanted mice were analyzed
to gain insight into the CD8+ T cell populations that
mediate GVHD directed against multiple miHA disparities. The TDL pool
is an excellent site for monitoring T cell responses since recently
activated T cells expand and enter the thoracic duct after initially
encountering miHA in the spleen and lymph nodes. Highly viable (99%)
cells can be retrieved from the TDL pool, containing significant levels
of blast-like T cells, in contrast to retrieval of T cells from the
peripheral lymphoid organs (28). Due to the overall low frequency
responses involved in anti-miHA reactivity and the slower
development of primary CD8+ T cell responses in vivo
relative to CD4+ T cells, significant phenotypic skewing of
Vß families was not detectable over a period of 3 to 8 days
posttransplantation of naive B6 T cells, although significant skewing
occurred in the CD4+ T cell population (18). The
cannulation of mice at later time points was impractical, since the
mice began developing GVHD-related symptoms and could not survive the
procedure. As an alternative, anti-host miHA T cells were first
expanded by presensitization and boosting of donor B6 mice in vivo with
host-type splenocytes. We then investigated the response patterns of
these donor cells when placed in a GVHD-inducing environment in the
irradiated BALB.B or CXBE recipients. To confirm that the TDL contained
alloreactive anti-miHA-specific T cells, TDL collected from the
B6 BALB.B combination were injected into lethally irradiated BALB.B
and CXBE recipients. The ability of these cells to induce GVHD in both
hosts suggested that the TDL contained alloreactive CD8+ T
cells. Lethal GVHD in the B6BALB.B model is mediated by either
CD4+ or CD4-dependent CD8+ T cells, while in
the B6CXBE model only CD4-dependent CD8+ T cells are
responsible for pathogenesis (17). The phenotypic analysis was not
sensitive enough to detect any major shifts in the B6BALB.B TDL but
did indicate expansions of at least the Vß10 and Vß14 families in
the B6CXBE TDL (Fig. 1
). This observation was consistent with the
involvement of a limited number of distinct miHA epitopes in the
development of the B6 CD8+ anti-CXBE GVHD response.
It was not overly surprising that phenotypic analysis of the
B6BALB.B TDL failed to detect a specific Vß family response. This
approach is capable of detecting only major shifts in Vß utilization,
and even if successful, it is unable to further characterize the
response and determine whether it is oligoclonal. A phenotypic increase
in Vß utilization could also be due to polyclonal expansion which
would represent increased expression of many TCR V-D-J sequences within
that Vß family. For example, polyclonal expansion was previously
observed for the CD4+ Vß3 family in the B6BALB.B
strain combination and was likely due to MTV-6 superantigen stimulation
(18). T cell repertoire analysis based on the size heterogeneity of the
CDR3 region is, therefore, a much more sensitive and powerful tool for
the study of GVHD and other types of immune responses (21, 29, 30). It
also provides a mechanism for differentiating the specific TCR V-D-J
sequences expressed within the same Vß family, and can reveal much
more subtle skewing within a Vß family than can be detected by FACS
analysis.
The increased Vß heterogeneity observed in the CDR3 size
spectratyping analysis would suggest that GVHD is mediated by a
heterogeneous population of alloreactive CD8+ T cells that
recognize either the same miHA, several miHA, or multiple epitopes of
the same Ag in the host. Yet, the overall responses to miHA in the
B6BALB.B and B6CXBE strain combinations were limited to only a
few Vß families, supporting the notion that a limited number of miHA
or epitopes were being recognized. The involvement of a limited number
of immunodominant miHA in GVHD has been previously found by Perreault
et al. in another model system (31). In addition, expansion of a
limited set of CD8+ Vß families in local liver GVHD
pathogenesis has been observed by Howell et al. (32). It has also been
shown that Ag-specific CD8+ T cells exhibit limited
heterogeneity at the level of TCR Vß gene usage during a primary
response to HIV infection (33). The notion of heterogeneous Vß
responses developing against a single or limited number of miHA is
highly possible since T cell recognition of Ag involves the interaction
of both Vß and V
chains (34). Each of the responding Vß chains
could associate with a different V chain to recognize the same
epitope. This has been observed in the diversity of CD4+ T
cell responses to influenza antigenic determinants (35).
It is estimated that more than 29 miHA locus differences exist between the B6 and BALB/c (and therefore the BALB.B) inbred mouse strains (36). Yet only a few immunodominant miHA appear to be recognized in vivo by MHC-matched allogeneic T cells responsible for skin graft rejection (37). An immunodominant hierarchy was also found to operate for the generation of miHA-specific CTL in vitro (14) and was possibly due to the comparative abilities of miHA to compete successfully for Ag-binding residues in the appropriate MHC molecules presented by APC. The affinity/avidity of the interaction of the specific TCR for miHA/self-MHC may also be an important factor of miHA immunodominance. Alternatively, T cells that respond more vigorously to strong miHA may down-regulate weaker responses to other miHA via cytokine production. Each of these potential mechanisms or combinations thereof could account for the phenomenon of competitive immunodominance. Recent studies comparing CTL immunodominant specificities and skin graft rejection in the B6 anti-BALB.B and CXB strain combinations have found a distinct lack of correlation between the two responses, with CTL recognizing only a limited number of miHA operative in vivo (13).
A similar lack of correlation with CTL immunodominant specificities had previously been found in GVHD (15, 17). For GVHD then, the relevant question becomes how extensive are the number of miHA that are actually involved in GVHD development? There must be certain limitations and qualifications for miHA to serve in this capacity. At least two of these criteria would seem to be immunogenicity and tissue distribution. In terms of the latter, it is known that some miHA have unique tissue expression (38, 39), and it would be expected that for optimum GVHD pathogenesis, Ag should be available not only in cells of hemopoietic origin but also in the primary target organs, including the intestine, liver, and skin. The lack of GVHD in irradiation chimeric models that have miHA expressed only in the host hemopoietic compartment supports the notion that the presence of miHA in both locations is required (40).
Thus, there may only be a limited number of miHA that fit the criteria
for GVHD. In this regard a recent study of the CXB interstrain lethal
GVHD responses suggested the minimal involvement of two distinct
immunodominant miHA (or associated groups of miHA) in the B6 BALB.B
GVHD response (16). One class I-restricted miHA (GVH-1) appeared to be
shared by the CXBE strain, while the second Ag (GVH-2) was uniquely
expressed by the BALB.B strain. The biased CDR3-size skewing of Vß1,
6, 8, 9, 10, and 14 families in both the BALB.B and CXBE recipients may
represent a response to common miHAs shared by BALB.B and CXBE mice,
i.e., GVH-1. Although the same CDR3 size bands were skewed in each
corresponding Vß family (except for Vß14), the final identity of
these specificities will depend on sequence analysis of the CDR3
segments involved in each response. Furthermore, the biased CDR3 usage
of Vß4 in the BALB.B recipients, not present in the spectratype of
the CXBE recipients, might represent a response to the unique BALB.B
miHA, i.e., GVH-2. Experiments are under way to determine whether
positively selected CD8+Vß4+ T cells will
induce GVHD in BALB.B but not CXBE recipients.
The biased CDR3 size skewing of Vß14 displayed the only nonidentical
skewing pattern between the B6BALB.B and B6CXBE T cell responses.
The BALB.B spectratype exhibited two additional skewed bands. The
additional bands present in the Vß14 spectratype of the BALB.B
recipients might also represent a response to the unique BALB.B miHA
(GVH-2). Although B6 Vß14+ T cells injected into either
BALB.B or CXBE recipients induced lethal GVHD (80% mortality by day 80
for both groups of recipients; Figs. 4B and
5B), the number of cells injected into the BALB.B
recipients was 30-fold less (1 x 105 vs 3.3 x
106) than into the CXBE recipients. This difference in the
response suggested a possible additive effect of different
anti-miHA clonotypes in the BALB.B, consistent with GVH-1 and
GVH-2. The capacity of presensitized Vß14+ donor T cells
to cause lethal GVHD is noteworthy on its own accord. It has been very
difficult in the past to demonstrate GVHD induction with normal donor T
cells directed to limited miHA specificities. The use of miHA congenic
strain combinations has failed to generate detectable GVHD, even when
donor cells were presensitized to host Ags (16, 41). Single cloned T
cells have been used successfully before to induce GVHD (42), but these
were CD4+ T cells. Furthermore, any clone (CD4+
or CD8+), due to their extensive expansion in culture,
could potentially differ from normal T cells in mediation of disease
pathogenesis (e.g., by abnormal acquisition or loss of adhesion
molecules responsible for migration into target tissue). The use of
normal donor T cells with limited heterogeneity to generate GVHD, as
with the Vß14+ cells, will ultimately enable the
investigation of associations between specific anti-miHA responses
and tissue distribution of lesions.
Characterization of the TCR usage by T cells responding to miHA and mediating GVHD will help us to understand the scope of these complex responses and ultimately the nature of the miHA responsible for their induction. With future developments in diagnostic capabilities, our appreciation for the GVHD response repertoires may also eventually allow for new targeted strategies for prevention of GVHD.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert Korngold, Kimmel Cancer Institute, Jefferson Medical College, 233 S. 10th Street, Philadelphia, PA 19107.
3 Abbreviations used in this paper: BMT, bone marrow transplantation; ATBM, anti-Thy-1 mAb plus C-treated bone marrow; B6, C57BL/6By; GVHD, graft-vs-host disease; MST, median survival time; miHA, minor histocompatibility antigen/s; RI, recombinant inbred; TDL, thoracic duct lymphocytes; CDR3, complementarity-determining region 3, DEPC, diethyl pyrocarbonate.
Received for publication September 18, 1997. Accepted for publication February 26, 1998.
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