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,
Departments of
*
Surgery and
Pathology, University of Wisconsin, Madison, WI 53792; and
Pathology and Laboratory Medicine Service, William S. Middleton Veterans Affairs Hospital, Madison, WI 53792
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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genes and made clonotype-specific probes
that allowed us to detect each clone in peripheral blood or biopsy
specimens obtained during a 1-year period before and after the rapid
onset of chronic rejection. We found an unexpectedly high level of
donor HLA-specific T cell clonotype mRNA in peripheral blood during the
late tolerance phase. Strong signals for two CD4+
clonotypes were detected in association with focal T cell infiltrates
in the biopsy. Chronic rejection was associated with a reduction in
direct pathway T cell clonotype mRNA in peripheral blood and the graft.
Our data are inconsistent with the hypothesis that direct pathway T
cells are involved only in early acute rejection events and suggest the
possibility that some such T cells may contribute to the maintenance of
peripheral tolerance to an allograft. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because of the availability of APC from both host and donor, the transplanted organ elicits different types of alloreactive T cells that recognize alloantigen through two distinct pathways: 1) indirect pathway, whereby soluble HLA or other donor-derived Ags are reprocessed and presented as antigenic peptides to host T cells that are restricted by "self" HLA proteins; and 2) direct pathway, whereby donor APCs within the graft or in the peripheral lymphoid tissues present the intact cell surface-bound allogeneic HLA plus bound peptide to recipient T cells. The classical view of the direct pathway T cells is that these high frequency responders are important only in the early posttransplant period, when donor dendritic cells migrate from the graft to the regional lymph nodes and spleen of the host. The host alloreactive T cells then become activated by contact with donor passenger leukocytes and migrate to the graft from the regional lymph nodes, where they mediate cellular acute rejection. Once the early acute rejections have been reversed by immunosuppressive drugs, and as host APC replace donor APC within the graft direct pathway T cells are thought to be replaced by indirect pathway T cells as principal mediators of late, chronic rejection (3). An alternative view is that under circumstances in which acute rejection has resolved, these direct pathway T cells may develop immunoregulatory functions (4). Thus, direct pathway T cells could either 1) be eliminated, 2) remain, but in a quiescent state, or 3) become dominant in a suppressive role during transplant tolerance. In either case, functional assays that rely upon growth and/or long term culture in presence of IL-2 may be unable to detect the donor-specific T cells that predominate in vivo (5).
For this reason, we chose to use a molecular approach to tracking
donor-specific T cells in vivo. To find a single clonotype with
particular Ag specificity, PCR-based tracking of T cells using TCR
junctional (V-D-Jß and V-J) sequence-specific oligo primers or
sequence-specific probes has been developed and utilized for the
analysis in patients with heart transplants (6), multiple
sclerosis (7), and HIV infection (8). Nixon
et al. (8) compared two clonotype analysis methods, one
using mRNA and RT-PCR and the other using genomic DNA and direct PCR.
They found that although the results were similar, they were not always
identical; and the authors concluded that whereas DNA-based analysis
reflected only the number of clones present in a sample, mRNA RT-PCR
signals represent not only the number of
clonotype+ T cells but also their activation
state. Although mRNA signals for TCR are not necessarily parallel to
the frequency of the T cells, the sensitivity of the RT-PCR clonotype
assays to detect Ag-specific T cells is much higher than that of
traditional in vitro assays such as CTL precursor or Th lymphocyte
precursor assay (7). In addition, unlike traditional
functional tests, clonotype analysis can easily be applied to biopsy
samples, so that graft-infiltrating alloreactive T cell frequency can
be estimated without resorting to culture techniques that may select a
new repertoire.
We analyzed mRNA samples from the graft and peripheral blood of a renal allograft recipient between 8 and 10 years posttransplant during the transition from functional tolerance to chronic rejection, and we measured the RT-PCR signals of direct pathway T cell clones specific for allo-HLA Ags expressed on donor cells. The data suggest that direct pathway CD8+ T cell clones may be readily detected by clonotype analysis even when such T cells cannot be detected by conventional precursor frequency analysis (2). The data also suggest that maintenance of peripheral tolerance in the absence of immunosuppressive drug therapy is compatible with high levels of direct pathway allospecific CD4+ T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The clinical status of the patient was monitored by serum creatinine level and by periodic biopsy studies. Biopsy sections were stained with hematoxylin and eosin and graded according the Banff criteria (9).
PBL and lymphoblastoid cell lines
PBL were obtained from patient JB (2), from his mother CL (the kidney donor), and from HLA-typed third-party individuals. B-lymphoblastoid cell lines (B-LCL)3 from patient, donor, and third parties were generated using EBV transformation of B cells. All PBL were initially typed for HLA-A, -B, -DR, and -DQ using microcytotoxicity (10). Subsequently, HLA-DR subtype assignments were made using PCR and sequence-specific oligonucleotide probes (11), and these assignments were confirmed by DNA sequencing.
Generation of CTL lines and clones
PBL obtained from the patient were cultured with gamma-irradiated donor PBL in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with either 10% patient’s plasma or FCS, penicillin, and streptomycin for 7–10 days (primary MLC). Patient’s PBL were restimulated with gamma-irradiated donor PBL in the presence or absence of 10 U/ml rIL-2 for 7 days (secondary MLC). T cell clones were then generated by limiting dilution culture at 0.9–90 cells/well in the presence of rIL-2. In the experiment leading to the derivation of clone JD6, gamma-irradiated fresh autologous cells containing donor-derived veto cells (2) were used as a feeder layer in combination with gamma-irradiated donor (CL) stimulator cells. In all other experiments, clones and lines were derived by limiting dilution in the presence of gamma-irradiated donor PBL alone. To generate CD8+ T cell lines, CD4+ T cells were depleted by addition of anti-CD4 mAb (Becton Dickinson, Mountain View, CA) followed by goat anti-mouse IgG-coupled immunomagnetic bead (Dynal, Lake Success, NY) selection as described previously (2). Limiting dilution culture was the same as for CD4+ T cell cloning, except that, in addition to donor gamma-irradiated PBL stimulator cells, 5 µg/ml PHA-P (Sigma, St. Louis, MO) was added only at the initiation of culture, and expanded clones were maintained without PHA. From 4 to 6 wk after limiting dilution, both CD4+ and CD8+ clones were screened by CTL assay as described previously (2) using B-LCL targets.
TCR Vß and V analyses by PCR
Total RNA was isolated from cell lines or clones (1 x
106 cells) by using the GLASSMAX RNA
Microisolation Spin Cartridge System (Life Technologies, Gaithersburg,
MD). RNA (1 µg ) was reverse-transcribed into first-strand cDNA by
using random hexamer primers (Life Technologies) according to the
manufacturer’s instruction. The cDNA was amplified with 19
Vß-specific (12) or 22 V-specific (Clontech
Laboratories, Palo Alto, CA) oligonucleotides. The PCR conditions were
denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and
extension at 72°C for 2 min, for 36 cycles on a thermal cycler (DNA
Thermal Cycler, Perkin-Elmer, Foster City, CA). The PCR products were
electrophoresed on 2% agarose gels, and the electrophoresed PCR
products were transferred to a nylon Zeta-Probe membrane (Bio-Rad,
Hercules, CA). After Southern blotting using radiolabeled either Cß
probe (5'-TTCTGATGGCTCAAACACAGCGACCTCGGG-3') or C probe
(5'-GTGTACCAGCTGAGAGACTCTA-3'), bands were visualized by
autoradiography on x-ray films.
DNA sequencing
TCR V and Vß gene sequencing of each clone was performed
using corresponding TCR V or Vß primers with either C or Cß
sequencing primers (C primer = 5'-TCTCAGCTGGTACACGGC-3'; Cß
primer = 5'-CTCAAAACACAGCGACCT-3'). PCR products were purified
using the Magic PCR Preps DNA Purification System (Promega, Madison,
WI) according to the manufacturer’s instruction. The purified PCR
products were sequenced by the Nucleic Acid Facility (Iowa State
University, Ames, IA), using the sequencing primers.
Clonotype analysis of PBL and kidney biopsy
For semiquantitative clonotype analysis, we utilized modified methods developed by Hu et al. (6). Five hundred nanograms of RNA from PBL or kidney biopsy were reverse-transcribed into cDNA as described. The cDNA was amplified by PCR for 36- 39 cycles using corresponding Vß primer to each clonotype. Oligonucleotides complimentary to the clone-specific junctional region were used for Southern blotting (JD6 probe 5'-TCCCCGGACCCTGTCAGGAA-3', F3 probe 5'-CTGGGGCTGCACCCTGTC-3', 7.22 probe 5'-CCGGGGGTCCCGTCCAACTG-3', 7.19 probe 5'-TCCAGTGATCTGTCGTAAGC-3', 9.21 probe 5'-TAGGGGAACCTGTCCCGGGG-3', 10.7 probe 5'-GCCATAGTTGGGGATTCG-3') The sensitivity of the clonotype detection assay ranged from 0.0002% to 0.1% in most experiments. The density of bands on autoradiograms was linearly correlated with the clonotype number in the range between a clonal frequency of 10-5 and 10-3. In all experiments, the density of positive blots obtained from PBL was less than the density obtained at a frequency of 10-3 in the standard. To assure the quality of each cDNA and to control for the quantity of TCR ß-chain expressed in each blood or tissue sample, control PCR was conducted using primers for GAPDH (5'-CCATGGAGAAGGCTGGGG-3', 5'-CAAAGTTGTCATGGATGACC-3') and Cß (5'-GAGGACCTGAACAAGGTG-3', 5'-CATTCACCCACCAGCTCAGCT-3'). The PCR products were stained by ethidium bromide after electrophoresis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As we reported previously (2, 13), patient JB with
end stage focal sclerosing glomerulonephritis received a kidney from
his mother (CL) after 3 donor-specific blood transfusions (DST) under
azathioprine coverage. Table I
shows HLA
haplotypes of the individuals used in the study, indicating the
noninherited maternal Ag (NIMA) HLA with underline. Interestingly, the
patient and maternal donor were serologically HLA-DR identical (DR4,5);
on DNA typing, the NIMA HLA-DR5 was found to differ in subtype
(DR5/11041) from the paternal inherited HLA-DR5/1102 allele, a
difference of only 2 amino acids in the DRß polymorphic domain
(14). Fig. 1 shows the
clinical course and serum creatinine levels of the patient. JB
experienced two reversible acute rejection episodes within 1 month
after the transplantation (2) and none thereafter. Of the
patient’s own volition, cyclosporin A therapy was discontinued after 3
mo, and the remaining steroids and azathioprine were stopped 2 years
posttransplantation at which time a biopsy (Biopsy 1) was performed. As
we previously reported (13), the biopsy at yr 2.2 showed
intense focal mononuclear cell infiltration adjacent to an area of
fibrosis and arteriolar intimal hyperplasia, but glomeruli and tubuli
were normal without cellular infiltrates. To resolve persistent
hypertension and proteinuria, which had resulted in a rise in serum
creatinine at yr 2.5, bilateral native nephrectomy was performed at 3.0
yr posttransplantation (Fig. 1). Thereafter the graft retained good
function until 9.0 yr posttransplantation, as evidenced by serum
creatinine < 2.0 mg/dl. Due to a slight rise in serum creatinine
to 2.1 mg/dl, a second biopsy was performed at yr 9.1. No evidence of
chronic rejection was present at this time. Instead, the biopsy
histology showed focal mononuclear infiltrates (FMI) and interstitial
fibrosis (Fig. 2B). Although
the size of focal infiltrates was larger than that in the earlier
biopsy (13), no cellular infiltration in either tubules
(T) or glomerulus (G) was observed (Fig. 2A). A rise from
2.1 to 9.8 mg/dl in serum creatinine occurred between yr 9.2 and yr
10.7, and a third biopsy at yr 9.7 confirmed the diagnosis of chronic
rejection as the cause of graft loss. Diffuse mononuclear infiltrates
(DMI) instead of focal infiltrates were observed in the third biopsy
specimen (Fig. 2D). Diffuse interstitial fibrosis, tubular
atrophy, arterial obstruction, and glomerular edema/fibrosis were
manifested (Fig. 2C, Banff criteria: chronic allograft
nephropathy grade III).
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We have previously described, using conventional CTL precursor
frequency analysis, a functional unresponsiveness toward donor HLA
class I Ags in this patient during stable tolerance (yr 4–6). The
unresponsive state was found to be associated with presence of rare
donor-derived cells in peripheral blood capable of inhibiting CTL
propagation in vitro (2). CTL anergy was profound,
requiring at least two rounds of stimulation with donor cells and
exogenous IL-2 to restore specific CTL function. Thus the use of CTL
functional assays to monitor CD8+ donor-specific
T cells during tolerance was not practical. Furthermore, functional
assays of the donor-specific CD4+ T cells in the
allograft was not practical, given the low yield of infiltrating cells
and requirement for in vitro culture. Therefore, to investigate the
role of direct pathway T cells specific for donor HLA in the change
from stable tolerance to chronic rejection, we used a strategy based on
analysis of anti-donor T cell clones established from limiting
dilution culture of peripheral blood T lymphocytes during the stable
tolerance phase (yr 6–7 posttransplant (Fig. 1
)). Because each T cell
clone manifested some degree of cytotoxicity toward donor target cells
after prolonged in vitro culture, we used a panel of B-LCL targets that
allowed us to dissect reactivity to individual donor mismatched HLA
(NIMA) to determine the specificity of each clone. As shown in Table II, two representative
CD4+CD8- clones (F3, 7.22)
and one
CD4+CD8+CD8ß-
clone (JD6) (15) recognized only target that expressed the
donor-subtype HLA-DR5/1104 alloantigen (HLA-DRB1*11041) or the LG
target which had HLA-DR5/1101 (DRB1*11011) and 1103 (DRB1*1103) Ags
(see Tables I and II). The donor HLA-DRB1*1104 subtype differs from
recipient DRB1*1102 in 2 amino acids at positions 67 and 71 adjacent to
the peptide-binding groove (Fig. 3). The
DRB1*1101 subtype shares the ß67F/ß71R motif with DRB1*1104 subtype
but differs at position 86 from both DRB1*1104 and DRB1*1102, whereas
the DRB1*1103 subtype shares ß67F and ß86V with donor DRB1*1104 but
has the same ß71E residue as the recipient 1102 Ag (Fig. 3).
Interestingly, it has been reported that DR1101 and DR1104 had high
cross-reactivity with each other in T cell recognition assays, less
with DR1103 and the least or almost none with DR1102 (16, 17).
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). Both of the CD8+
clones, 9.21 and 10.7, recognized only target cells which expressed
HLA-B62, suggesting that these, like CD4+ clones
F3, 7.22, and JD6, were also donor-HLA-specific, direct pathway T
cells. TCR gene sequences of donor-reactive CD4+ and CD8+ clones
We determined the Vß and V assignments of the clones by
RT-PCR using a panel of Vß and V primers, followed by Southern
blot analysis using a Cß or C-specific probe. To determine whether
T cell clones having similar allospecificity were characterized by a
common sequence motif and junctional (complementarity-determining
region 3) sequence homologies, we sequenced the expressed TCR ß genes
of CD4+ and CD8+ clones.
The TCR genes of the CD4+ clones were also
sequenced. The nucleotide and predicted protein sequences spanning the
junctional VDJ regions of the TCR ß-chains of the clones are shown in
Fig. 4. The sequence analysis revealed no
common junctional sequence homologies in the expressed V and ß of
the HLA-DR5/1104-specific T cell clones. CD4+
clones JD6, F3, and 7.22 each used different Jß families (BJ2S2,
BJ1S5, and BJ1S3, respectively) and differed in the length of N + Dß
+ Jß region (Fig. 4A); each clone also used different TCR
V chains (AV8S2, AV2S3–5, and AV16S1, respectively) (Fig. 4A) and different J genes (data not shown). A similar
high degree of TCR V gene diversity in alloreactive
CD4+ T cell clones raised against a closely
related HLA-DR Ag has been reported previously (18).
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B). Although clones
9.21 and 10.7 both lysed only HLA-B62+ target
cells, each used different Vß (BV9S1 and BV13S2) and Jß (BJ2S7 and
BJ1S2) and had no common junctional VDJ amino acid sequences.
Autoreactive CD4+ clone 7.19, which lysed both
donor and autologous LCL (but not all LCL in the panel), expressed
Vß1, and, as expected, had no homology to either
CD4+ or CD8+ direct pathway
clones in its TCR gene sequences (Fig. 4C). Clonotype analysis of anti-donor T cells in PBL before and after the onset of chronic rejection
The unique junctional sequences present in each clone (Fig. 4, underlined sequences) enabled us to develop specific oligonucleotide
probes for clonotype detection analysis. Fig. 5A shows an example of assay
sensitivity. A varying number of Vß8+ JD6
clones were mixed into donor CL PBL (negative control) to a total of
106 cells. Five hundred nanograms of extracted
RNA from each sample were reverse transcribed and used as a template
for PCR with Vß8 primers, and clonotype was detected using
radiolabeled JD6 junctional TCR ß probe. Two positive cells in
106 cells were detectable after a 39-cycle PCR
(Fig. 5A).
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B, densities of mRNA
signals of TCR genes in the direct pathway CD4+ T
cells (F3, JD6, and 7.22) showed a similar pattern: all three
clonotypes were readily detected in samples obtained at yr 8.8 and yr
9.1 (pre-chronic rejection, late tolerance phase) with clonotypes F3
and 7.22 giving the strongest signals. The density of mRNA signals from
all three CD4+ direct pathway clonotypes (JD6,
7.22, and F3) was decreased at 9.2 yr posttransplantation and around
the time of biopsy-proved chronic rejection (9.7 and 9.8 yr). Although
clonotype F3 remained detectable in PBL, clonotype 7.22 and JD6 mRNA
signals fell to background level. Based on the standard curve for JD6,
there was at least a 10-fold reduction in clonotype mRNA between yr 9.1
and yr 9.2, i.e., within a period of <2 mo after biopsy 2.
The intensity of one of the donor class I-specific
CD8+ clonotypes, 9.21, changed in the same manner
as the CD4+ anti-donor class II-specific
clonotypes, 7.22 and F3, being strongly expressed at both prerejection
time points, decreasing sharply with onset of chronic rejection. Like
F3, the 9.21 mRNA signal in PBL persisted during chronic rejection but
at a reduced level relative to yr 8.8 and 9.1 samples. This finding was
in contrast to functional studies of anti-donor CTL, which showed
no evidence of functional CTL in primary limiting dilution assay or
bulk culture analysis during the early or late tolerance phase (Ref.
2 ; S. Kusaka, A. P. Grailer, and W. J.
Burlingham, unpublished observations). This suggests that clonotype
analysis, like HLA-tetramer analysis (19), can reveal the
presence of anergic CD8+ T cells. The other
CD8+ clonotype, 10.7, was barely detectable at
all time points. The loss of mRNA signals for 4 of 5 anti-donor
direct pathway clonotypes was well correlated with the increase of
serum creatinine during the same period (Fig. 2). Interestingly, the
signal of the one autoreactive CD4+ T cell
clonotype, 7.19, was detected only at yr 9.1.
To rule out the possibility that levels of clonotype detected using
oligonucleotide probes for TCR ß only might reflect
non-donor-specific T cells due to pairing with irrelevant TCR , we
analyzed the expression of TCR mRNA signals. A similar pattern of
loss of the mRNA signals between yr 9.1 and 9.2 was confirmed by a
similar method using primers for V16 and V2, and TCR
junctional gene sequence-specific probes for 7.22 and F3 (data not
shown). As shown in Fig. 5B, the densities of both Cß and
GAPDH signals were equivalent between each time point, indicating that
the quality of cDNA obtained at each time point was good and the number
of T cells were comparable. This suggests that the observed loss of TCR
Vß mRNA signals from direct pathway clonotypes was not an artifact of
general loss of T cells or T cell mRNA signals.
Analysis of alloreactive clonotype-positive T cells in the graft
To determine whether the loss of mRNA signals of anti-donor
direct pathway T cells from peripheral blood was the result of a
redistribution of those clonotypes from the peripheral blood into the
graft during chronic rejection, we performed clonotype analysis of
biopsy specimens collected pre- (yr 9.1) and post- (yr 9.8) chronic
rejection for all six direct pathway allospecific clonotypes (Fig. 6). Of the six clonotypes that were
detected in the PBL at the prerejection time points, mRNA signals of
only clonotypes 7.22 and F3 were readily detectable within the graft at
yr 9.1, the prerejection time point. Clonotypes JD6 and 9.21 did not
redistribute to the graft site after the onset of rejection, remaining
undetectable in the biopsy mRNA (data not shown). Of the two clonotypes
that were present in the graft at yr 9.1, the mRNA signal for the F3
clonotype was completely lost from the graft after chronic rejection,
whereas 7.22 clonotype mRNA was still detectable but at a lower signal
intensity (Fig. 6). Both Cß and GAPDH mRNA signals in the biopsy
samples were unchanged between the pre- and postrejection time points
as measured by intensity of ethidium bromide staining of the
appropriate RT-PCR products. Furthermore, analysis of RT-PCR products
from the same mRNA samples using an HPLC semiquantitative method also
indicated that similar amounts of TCR ß mRNA were present in pre- and
postrejection biopsy samples (20). Thus, the loss of mRNA
signals for direct pathway T cell clonotype, F3, JD6, and 9.21 in the
peripheral blood was not the result of a redistribution of these clones
to the graft. The loss of mRNA signals for clonotype 7.22 from the
peripheral blood, in contrast, may have been due in part to such a
redistribution, because it was detected weakly in the graft but not in
the blood during chronic rejection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The
cross-reactivity among these 4 DRB1*11 alleles was precisely analyzed
using influenza HA306–320 peptide-specific (16) and
tetanus toxin peptide p2-specific (17) DR11-restricted
CD4+ T cell clones. These studies agreed that
most of the DR1101-restricted CD4+ T cell clones
recognized DR1104, and they had less reactivity with DR1103 and the
least or almost none with DR1102, even though all four DR11 types had
similar binding capacity of the same antigenic peptides. In other
words, the ß67F/ß71R motif which is located in the middle of the
Ag-binding groove (Fig. 3) (24) is the most critical in
the Ag recognition by CD4+ T cells. Whether those
CD4+ T cell clones recognized DR1101, DR1103, or
both was not clear, but they failed to recognize recipient DR1102
expressed by autologous LCL. Although we cannot rule out the
possibility that autologous LCL, when pulsed with the appropriate
allopeptides, could express the ligand for these clones, the common
recognition of DR1104- and DR1101/1103-positive targets argues that the
epitope is donor not host DR restricted. It appears likely that all
clones except autoreactive clone 7.19 recognized donor Ags through the
direct allorecognition pathway. We found a surprisingly high level of donor-specific CD4+-CD8+-T cell clonotypes in PBL and two CD4+ clonotypes in the graft during the late tolerance, prerejection stage using clonotype analysis. By comparing the pattern of donor HLA-specific T cell clonotype mRNA expression in the graft and peripheral blood before and after the onset of chronic rejection, and by examining other parameters such as renal histopathology and soluble donor HLA release, we found that the loss of the tolerance phenotype in patient JB was associated with a decrease rather than an increase in the RT-PCR signal for donor-specific direct pathway CD4+ T cell clonotypes isolated during the stable tolerance phase. This decrease in direct pathway T cell clonotype mRNA in PBL preceded increases in serum creatinine and in the level of soluble donor HLA Ag in serum (data not shown), indicating that changes in the alloreactive T cell repertoire may be a leading indicator of chronic rejection.
There are at least two possible interpretations of these findings. One is that direct pathway CD4+ and CD8+ T cells were quiescent during the stable tolerance phase (yr 2–8), but for unknown reasons they became activated just before the manifestation of chronic rejection (yr 8–9) and may themselves have triggered the rejection. Subsequent clonal exhaustion or activation-induced cell death, which are commonly seen in Th1 cells (25), could have resulted in a loss of clonotypes from the graft and the blood.
A second possibility is that the direct pathway
CD4+ and CD8+ T cells that
we cloned during the tolerance phase are ones that lack cytolytic
function in vivo and may in fact inhibit proinflammatory Th1 cells. The
unusual phenotype (CD4+
CD8+ß-) of many of
the direct pathway CD4+ T cell clones (including
JD6, for example) isolated from the peripheral blood during the
tolerance phase is consistent with this interpretation
(15). In this view the loss of these immunoregulatory T
cells parallels the onset of chronic rejection and is related to the
breakdown of tolerance.
The case for active immunoregulation of the anti-donor response in this patient is based on data from several sources. As noted previously, JB received three donor blood transfusions from his mother before the kidney transplant and displayed a relatively high level of microchimerism in peripheral blood and skin (2). It has been reported that graft passenger leukocytes, especially dendritic cells, may play an important role for graft acceptance in liver transplants (26) and DST-treated recipients (27). DST treatment has also long been known to induce suppressor cells (28, 29). Although the presence of such cells was controversial, recent reports have shown that they could be analogous to Tr-1 cells (30, 31) and that DST treatment preferentially induces graft-infiltrating cells that produce TGF-ß1, potential suppressor cells (32). Under conditions of microchimerism in which rare leukocytes of donor origin persist within the transplant recipient’s peripheral blood and tissues, donor APC may contribute both to the persistence of direct pathway T cells and to their control via immunoregulation (2). Another possible driving force for the induction of such suppressive cells in this patient was a priming by noninherited maternal HLA. Recent studies of neonatal Ag exposure suggest that the common result of Ag priming in utero is a memory response that is antagonistic to Th1 function (33, 34, 35, 36). By this interpretation, the loss of direct pathway CD4+ clonotype mRNA signals observed at the breakdown of tolerance might reflect a change in the longstanding (i.e., since in utero life) dominance of certain alloreactive memory T cell clonotypes, leading to reduced activation of direct pathway T cells that have immunoregulatory function (1). The reduced mRNA signals for clonotypic TCR between yr 9.1 and 9.7 could reflect a drop in clonal frequency or a decrease in activation, given that the synthesis of TCR mRNA may be linked to the cycling of the TCR after Ag-induced receptor modulation (8). Either way, the influence of the clonotypic T cells on events within the graft and the periphery would be diminished.
Our recent studies using the newly developed trans-vivo delayed-type
hypersensitivity (DTH) assay (37) showed that this
patient’s PBL from tolerant phase (yr 8.8) had no DTH reaction against
a donor alloantigen sonicate preparation and that PBL after the
rejection caused high DTH reaction against a donor Ag sonicate. These
data suggest that host T cell-mediated reactivity to donor Ag through
indirect pathway was only manifested after the breakdown of tolerance
(W. J. Burlingham, E. Jankowska-Gan, A. M. VanBuskirk, and
C. G. Orosz, manuscript in preparation). The timing of this
increase of DTH reaction was coincident with the sharp rise in serum
creatinine and donor soluble HLA class I level between yr 9.2 and yr
9.7. Interestingly, the rapid decline in direct pathway T cell
clonotype transcripts in PBL was seen 1 mo earlier than the rise of
creatinine (between yr 9.1 and yr 9.2 (Fig. 5)). During this same 1-mo
interval, the serum creatinine level remained low, suggesting that a
decrease in the direct pathway T cells, like the increase in indirect
pathway DTH reactivity, may have preceded the renal damage that gave
rise to increased serum creatinine level.
In conclusion, T cell clonotype analysis of peripheral blood and graft biopsy appears to be a useful approach for the study of human allograft tolerance. Our data suggest that the direct pathway of allorecognition of donor HLA Ags, including both CD4+ and CD8+ T cell clones, persisted in the period of tolerance despite the absence of CTL function and the presence of inhibitory donor-derived leukocytes (2). The loss of these clonotypes occurred before a marked increase in serum creatinine and was associated with a change from a focal, interstitial to an invasive graft T cell infiltrate. Whether or not the direct pathway T cell clones derived from the peripheral blood during the stable phase of tolerance plays a positive role in the stabilization of the tolerant state remains to be determined. The clonotype analysis approach, as well as other direct approaches to quantitation of Ag-specific T cells in vivo including HLA dimer (38) and tetramer strategies (39) may therefore open a new avenue to analyzing donor-reactive T cells during peripheral tolerance, when conventional functional in vitro assays may fail to detect their presence.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Satoshi Kusaka, Department of Surgery, University of Wisconsin Hospital, 600 Highland Avenue H4/781 CSC, Madison, WI 53792.
3 Abbreviations used in this paper: B-LCL, B lymphoblastoid cells; CTLp, cytotoxic T lymphocyte precursor; DST, donor-specific blood transfusion; HTLp, helper T lymphocyte precursor; NIMA, noninherited maternal Ag; DTH, delayed-type hypersensitivity.
Received for publication July 8, 1999. Accepted for publication December 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helix motif shared by DR11 and DR8 alleles is implicated in the pluriallelic restriction of peptide-specific T-cell lines. Hum. Immunol. 40:279.[Medline]
and TGFß1 genotype: partial association with intragraft
gene expression in two cases of long term peripheral tolerance to a
kidney transplant. Transplantation 69, In press.
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  A. Miyagawa-Hayashino, T. Tsuruyama, H. Egawa, H. Haga, H. Sakashita, T. Okuno, S. Toyokuni, K. Tamaki, H. Yamabe, T. Manabe, et al. FasL Expression in Hepatic Antigen-Presenting Cells and Phagocytosis of Apoptotic T Cells by FasL+ Kupffer Cells Are Indicators of Rejection Activity in Human Liver Allografts Am. J. Pathol., November 1, 2007; 171(5): 1499 - 1508. [Abstract] [Full Text] [PDF]
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 A. D. Salama, K. L. Womer, and M. H. Sayegh Clinical Transplantation Tolerance: Many Rivers to Cross J. Immunol., May 1, 2007; 178(9): 5419 - 5423. [Abstract] [Full Text] [PDF]
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Q. Xu, J. Lee, E. Jankowska-Gan, J. Schultz, D. A. Roennburg, L. D. Haynes, S. Kusaka, H. W. Sollinger, S. J. Knechtle, A. M. VanBuskirk, et al. Human CD4+CD25low Adaptive T Regulatory Cells Suppress Delayed-Type Hypersensitivity during Transplant Tolerance J. Immunol., March 15, 2007; 178(6): 3983 - 3995. [Abstract] [Full Text] [PDF]
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 I. A. Laskowski, J. Pratschke, M. J. Wilhelm, V. M. Dong, F. Beato, M. Taal, M. Gasser, W. W. Hancock, M. H. Sayegh, and N. L. Tilney Anti-CD28 Monoclonal Antibody Therapy Prevents Chronic Rejection of Renal Allografts in Rats J. Am. Soc. Nephrol., February 1, 2002; 13(2): 519 - 527. [Abstract] [Full Text] [PDF]
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 P. A. Muraro, K.-P. Wandinger, B. Bielekova, B. Gran, A. Marques, U. Utz, H. F. McFarland, S. Jacobson, and R. Martin Molecular tracking of antigen-specific T cell clones in neurological immune-mediated disorders Brain, January 1, 2002; 126(1): 20 - 31. [Abstract] [Full Text] [PDF]
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R. J. Baker, M. P. Hernandez-Fuentes, P. A. Brookes, A. N. Chaudhry, H. T. Cook, and R. I. Lechler Loss of Direct and Maintenance of Indirect Alloresponses in Renal Allograft Recipients: Implications for the Pathogenesis of Chronic Allograft Nephropathy J. Immunol., December 15, 2001; 167(12): 7199 - 7206. [Abstract] [Full Text] [PDF]
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