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TRANSPLANTATION
From the Oncology Center, Johns Hopkins University
School of Medicine, Baltimore, MD; the Department of Hematology and
Immunology, Ishikawa Prefectural Central Hospital; and the Third
Department of Medicine, Kanazawa University School of Medicine, Japan.
Administration of cyclosporine A (CsA) after autologous stem cell
transplantation elicits an autoimmune syndrome with pathology similar
to graft-versus-host disease (GVHD). This syndrome, termed autologous
GVHD, is associated with the appearance of autoreactive T cells
directed at major histocompatibility class (MHC) class II antigens. In
the rat model of autologous GVHD, clonal analysis reveals that the
effector T cells are highly conserved and recognize a peptide from the
invariant chain peptide presented by MHC class II. Although human
autologous GVHD effector T cells share a similar phenotypic
specificity, clonality of the response in humans has not been
determined. To examine the human effector T-cell response, the T-cell
repertoire of peripheral blood lymphocytes was assessed by
complementarity-determining region 3 (CDR3) size distribution analysis
and T-cell clonotype analysis in 26 patients treated with CsA after
transplantation. Autologous GVHD developed in 3 of 4 patients with
human leukocyte antigen (HLA)-DRB1*0701, and clonal expansions of
High-dose chemoradiotherapy combined with
autologous stem cell transplantation (ASCT) has been used successfully
in the treatment of patients with malignant lymphoma or breast cancer.
Clinical studies have demonstrated that among the therapeutic options
available for relapsing lymphoma or metastatic breast cancer, ASCT is
the most effective in achieving long-term survival.1-4
However, clinical trials have failed to demonstrate any advantage of
ASCT over conventional chemotherapy in the treatment of patients with
either non-Hodgkin lymphoma (NHL) and a slow response to chemotherapy,
with disease in remission but poor prognostic factors,5,6
or breast cancer with extensive lymph node involvement.7
These studies suggest that raising the dose intensity of chemotherapy,
facilitated by the use of ASCT, may not necessarily improve the
outcome. For patients who do not achieve sustained remission after
conventional chemotherapy, novel approaches including immunotherapy
are needed.
One such approach is the induction of autologous graft-versus-host
disease (GVHD) after ASCT. Autologous GVHD is inducible in recipients
of autologous bone marrow by the administration of cyclosporine A (CsA)
for a short period after transplantation,8-13 resulting in
an autoaggression syndrome that has a dermal pathology similar to that
of acute GVHD after allogeneic marrow transplantation. Interestingly,
CD8+ T cells that develop during autologous GVHD are also
able to lyse myeloma and breast cancer cell lines.14,15
T-cell clones associated with autologous GVHD have been isolated in
rodents16,17 and are restricted to the T-cell receptor
(TCR) To evaluate this hypothesis, the T-cell repertoire in peripheral blood
lymphocytes (PBL) from 26 patients treated with CsA after ASCT was
analyzed. Complementarity-determining region 3 (CDR3) size distribution
analysis and sequencing of TCR cDNA were used to determine the
clonality of T-cell expansion for each BV family. The CDR3 size pattern
of several BV subfamilies that were normal before ASCT became abnormal
after ASCT plus CsA treatment, suggesting that clonal or oligoclonal
expansion of a limited number of T cells had occurred. Among these BV
subfamilies, clonal expansions within BV16+ T cells were
shared by 7 of 11 patients in whom autologous GVHD developed and in 5 of 15 patients without clinical evidence of GVHD. Interestingly, both
autologous GVHD and clonal expansion within BV16+ T cells
developed in 3 of 4 patients with human leukocyte antigen (HLA)-DRB1*0701. Moreover, clonal expansions within BV15+
or BV22+ T cells were detected in 4 of 6 patients with
HLA-DRB1*1501 and in 3 of 4 patients with HLA-DRB1*0401, respectively.
Sequencing of BV16 cDNA for which the CDR3 size profile exhibited
apparent clone predominance in autologous GVHD-induced patients
revealed significant homology in the joining segment. The same amino
acid sequence of the CDR3 domain occurred in 2 different patients with HLA-DRB1*0701 or HLA-DRB1*1502.
Patients and autologous blood cell transplantation
All patients with NHL (group A) underwent chemotherapy before
autologous peripheral blood stem cell transplantation (PBSCT). Before
PBSCT, 2 patients underwent conditioning with high-dose chemotherapy
consisting of ramimustine (200 mg/m2 intravenously
(IV) days CsA (2.5 mg/kg·d; Novartis, Hanover, NJ) was administered
intravenously to the patients from days 1 through 21 or 28. Recombinant interferon- Cytotoxicity assay Heparinized PB was collected from the patients before conditioning chemotherapy, and PBMCs were separated using density-gradient centrifugation, suspended in RPMI 1640 medium (Gibco, Grand Island, NY), and cryopreserved at 80°C. For testing
autocytotoxic activity, thawed PBMCs were cultured for 5 to 7 days in
the presence of 1 µg/mL phytohemagglutinin (Wellcome, Dartford,
United Kingdom). After washing with RPMI 1640, the
phytohemagglutinin-stimulated lymphoblasts (PHA blasts) were used as
targets in a chromium Cr 51 release assay. PHA blasts were suspended
for 1 hour in 10 µL 51Cr (400-1200 Ci/g chromium; Dupont
NEN Products, Boston, MA). After washing with phosphate-buffered
saline, 104 cells were cultured with 8 × 105
autologous PBMCs from each patient after ASCT. After 4 hours of
incubation, 51Cr release into the medium was measured using
a gamma counter. Percentage specific lysis (mean ± SE) was
determined from triplicate cultures as follows: 100 × (experimental
release cpm spontaneous release cpm)/(maximum release cpm spontaneous release cpm). To assess the specificity of the effector
T cells, the target cells were pretreated (1 hour at 4°C) with either
anti-MHC class II (anti-DR; Becton Dickinson) and anti-MHC class I
(DAKO, Carpinteria, CA) monoclonal antibodies or with antihuman CLIP
(kindly given by Dr Peter Cresswell). The anti-DR and anti-MHC class I
antibodies were used at a final concentration of 1:200. Target cells
were pretreated with 1.5 µg antihuman CLIP Fab preparation. After
antibody pretreatment, the cells were washed 3 times before assay.
RNA extraction and cDNA preparation Heparinized PB was collected from the patients after informed consent was obtained and PBMCs were separated using density-gradient centrifugation. Total RNA was extracted from PBMCs, and 1 µg RNA was reverse transcribed into cDNA in a reaction primed with oligo (dT) 12-18 by using SuperScript II reverse transcriptase, as recommended by the manufacturer (Gibco-BRL, Bethesda, MD).CDR3 size distribution analysis Conditions for the generation of CDR3 size distribution analysis have been reported elsewhere.22-24 Briefly, cDNA was polymerase chain reaction (PCR) amplified through 35 cycles (94°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute) with a primer specific to 24 different BV subfamilies (BVs1-20,25 BVs21-2423) and a fluorescent BC primer.25 Analysis of BV10 and BV19 was excluded from this study because these are pseudogenes.26 One microliter amplified products was mixed with 2 µL 100% formamide, heated at 90°C for 3 minutes and electrophoresed in a 6.75% denaturing polyacrylamide gel. The distribution of the CDR3 size within the amplified product of each BV subfamily was analyzed using an automatic sequencer (Applied Biosystems Division, Foster city, CA) equipped with the computer program Genescan Software (Applied Biosystems Division) allowing determination of the fluorescence intensity of each band. CDR3 size patterns that failed to exhibit a bell-shaped distribution owing to the appearance of prominent peaks with or without a reduced peak number (less than 5 peaks) were judged abnormal. This judgment was made by 3 different investigators to minimize interindividual differences.Cloning and sequencing of PCR-amplified cDNA The PCR products of BV16 or BV22 cDNA were cloned into a pCR II-TOPO Vector system (Invitrogen, Carlsbad, CA). Thirty colonies containing the insert fragment were randomly selected and sequenced using an ABI pRISM cycle Sequencing Kit (PerkinElmer, Norwalk, CT) and an automatic DNA sequencer (ABI 373; PerkinElmer). The amino acid sequence of the CDR3 region was deduced using the DNASIS-Mac v3.2 software (Hitachi Software, Yokohama, Japan).
Relation between autologous GVHD and HLA Clinical GVHD developed in 13 of 31 (41.9%) patients. Relations between certain HLA phenotypes and autoimmune diseases have been previously described.27 For example, the expression of HLA-DRB1*1501 or *0410, respectively, was found to be closely associated with susceptibility to aplastic anemia or idiopathic thrombocytopenic purpura.28,29 Therefore, the HLA phenotypes of 31 patients undergoing ASCT and receiving CsA to induce autologous GVHD were examined. Autologous GVHD developed in 3 of 4 patients with HLA-DRB1*0701.Cytotoxicity assay Autocytotoxic activity of lymphocytes obtained at different time points after ASCT was determined against autologous lymphocytes using the 51Cr release assay. Autocytotoxic activity of PBMCs was detectable in all patients treated with both CsA and IFN- . Figure
1 illustrates the profile of
autocytotoxic activity of lymphocytes obtained at different time points
after ASCT. Lymphocytes with autocytotoxic activity often appeared at
day 12, remained until day 35, and were no longer detected after day
36. Autocytotoxic activity peaked between days 15 and 28 and occurred
in a concentration-dependent manner.14,30 Such
autocytotoxic lymphocytes were never detected in ASCT patients who did
not receive CsA (Figure 1, patient 4). To determine whether the
cytotoxicity mediated by post-transplantation lymphocytes is restricted
by certain HLA molecules, blocking studies using monoclonal antibodies
were performed (Figure 2). Specific lysis
(20.4 ± 5.2; n = 3; mean ± SD) by the autocytotoxic effector T cells was blocked by the addition of anti-DR (7.4 ± 4.2) and anti-CLIP antibodies (4.3 ± 2.6), but not by anti-MHC class I antibody (19.4 ± 4.5).
CDR3 size distribution of TCRBV cDNA of PBL cDNA of 24 different BV subfamilies was amplified using a fluorescent BC primer, and the CDR3 size distribution of each BV subfamily was compared among healthy controls and the patients. The specificity of each T-cell was determined by the amino acid sequence of the TCR -chain CDR3 region, corresponding to diversity (D) region,
upstream and downstream nucleotides (N), and joining (J) region genes.
Diversity of CDR3 within the T-cell repertoire facilitates recognition
of a variety of antigens. CDR3 cDNA can be categorized in groups, each
differing by 3 nucleotides, constituting peptides from 6 to 12 amino
acids in length.22,31 Sequence analysis of electrophoretic
patterns that result from these groups has revealed that T-cell
subfamilies in which clonal expansion did not occur have a CDR3 size
distribution that was characteristically bell shaped (Figure
3A). In contrast, when clonal expansion
of a few T cells occurred, the bell-shaped distribution was skewed with
the appearance of a sharp peak (Figure 3C). All CDR3 size distribution
patterns from each BV subfamily in PBL from a healthy control and
Staphylococcus enterotoxin B (SEB) stimulated PBL from a
healthy control showed the normal pattern for polyclonality (data
not shown).
Figure 4 shows the CDR3 size distribution
pattern for each BV subfamily before ASCT (upper) and after ASCT
(lower) at the onset of autologous GVHD in patients 3 and 23 and in
another ASCT patient who did not receive CsA (control). In these
patients, abnormalities were found in the CDR3 size distribution
pattern for more than half of the subfamilies, most likely because of past chemotherapy and blood transfusion. On the other hand, at 26 days
after transplantation for the ASCT patient who did not receive CsA, all
subfamilies with skewed distributions before transplantation (upper)
had switched to polyclonal patterns, suggesting reconstitution of the
T-cell compartment (lower). Most subfamilies that gave a skewed
distribution before transplantation had changed from oligoclonality to
polyclonality, even in patients with autologous GVHD. However, this
analysis also revealed that the BV16 family and some other BV families,
which showed normal patterns before ASCT, developed CDR3 distribution
peaks consistent with monoclonal expansion. Although it cannot be
excluded that these abnormal CDR3 patterns were due to a delayed immune
reconstitution, these patterns did not occur in healthy controls or in
ASCT patients who were not administered CsA. Although the frequency of
BV families using anti-TCR BV monoclonal antibodies on an flow
cytometry system was compared between patients (patients 3 and 6),
immunofluorescent monoclonal antibody staining did not demonstrate
significant changes in the patients before or after transplantation
(data not shown). Previous studies indicate that clonotypic expansions
do not necessarily elevate the overall frequencies of TCR BV
families.32
Selection of TCR-
Deduced amino acid sequence of CDR3 region of BV16 cDNA These findings indicated that clonal expansion of a limited number of T cells is common in autologous GVHD-induced patients. To directly demonstrate the clonal expansion of a limited number of T cells, the amplified products of the BV16 cDNA derived from 10 patients (patients 6, 8, 12, 20, 22, 23, 24, 25, 28, 29) were cloned and sequenced. Table 3 summarizes the amino acid sequence of each clone. Interestingly, some patients exhibited a high frequency of clones with a specific N-D-N region RYRID (9/10) in patient 8, QVSRA
(9/19) in patient 12, RTGTTS in patient 20, AIGP (11/22) in patient 22, AYRDRRL (18/26) in patient 23, RIETYP (8/14) in patient 24, QDFPSGSWE
(7/19) in patient 25, AIGP (14/18) in patient 28, and HRGQG (9/15) in
patient 29. Such high frequencies of certain clones are compatible with
the results of CDR3 size distribution. Surprisingly, AIGP in patient 22 matches the sequence in patient 28 with the same DRB1*0701. Similarly,
the most frequent amino acid sequence, RYRID, in patient 6 occurred in
patient 8; both had the same HLA-DRB1*1502. All these clones expressed
a common TCR BJ element, BJ2S1. The CDR3 region interacts directly with the peptide epitope.33,34 These observations suggest that
at time of onset of autologous GVHD, clonal T-cell expansion against a
common antigen occurs in the PB. Of further interest was the observation of AYRDRRL (15/19) and RIETYP (8/14) in patients 23 and 24, respectively, who did not have clinical signs of autologous GVHD though
they received CsA after transplantation. Interestingly, these
high-frequency clones have the same codon (cgc tat or
ata) in the first and second portions of the hypervariable
CDR3 region (Table 4), a finding that is
unlikely to reflect selection by antigen.33,34 It appears
that clonal T-cell expansion occurred even in patients administered CsA
after transplantation, though they did not subsequently have clinical
autologous GVHD.
Relation between clonal expansion within certain BV family and HLA phenotype Autologous GVHD and clonal expansions within BV16+ T cells developed in 3 of 4 patients with HLA-DRB1*0701. Clonal expansions within BV15+ T cells were also shared by 4 of 6 patients with HLA-DRB1*1501 during the early post-transplantation period (days 12-19), and clonal expansions within BV22+ T cells were shared by 3 of 4 patients with HLA-DRB1*0401 during the late post-transplantation period (days 26-33). However, only one patient in each of these groups had clinical evidence of autologous GVHD. Although clonal expansions occurred within BV16, BV15, and BV22, only the expression of HLA-DRB1*0701 and not of HLA-DRB1*1501 or HLA-DRB1*0401 appears to be related to the development of autologous GVHD. Table 5 summarizes the amino acid sequence of TCR BV22 cDNA derived from PB of 5 patients (patients 11, 15, 18, 20, 25). Although high frequencies of certain clones were compatible with the results of CDR3 size distribution, identification of a common N-D-N amino acid sequence was not observed among 3 different patients with HLA-DRB1*0401 (patients 11, 18, 20).
The present analysis of T-cell repertoire in the PB of the patients with autologous GVHD reveals several new findings regarding the autoreactive T-cell repertoire in the pathophysiology of human autologous GVHD. Patients with autologous GVHD exhibited CDR3 size abnormalities suggestive of clonal predominance. These abnormalities did not appear in healthy controls or in ASCT patients not administered CsA. A subset of these changes overlaps the autocytotoxic activity (days 11-35), and skewed spectratype patterns of BV16 were recognized until day 33. This monoclonal T-cell expansion is in accordance with the appearance of autocytotoxic T cells. The abnormal CDR3 size patterns of patients cannot be attributed to a delay in the reconstitution of T cells because they were not detected in the ASCT patients not administered CsA. Furthermore, the skewed pattern cannot be detected on resolution of autologous GVHD beyond 56 days after transplantation. Thus, clonal expansion of a limited number of T cells in the PB appears to be related to the pathophysiology of autologous GVHD. Although the mechanism of autologous GVHD is unclear, recent studies
suggest that CD8+ effector lymphocytes recognize HLA-class
II determinants10-12 and that there is an infiltration of
BV15+ T cells in skin lesions.19 Additionally,
the molecule CLIP appears to play a major role as the target antigen in
autologous GVHD.17,18 CLIP is a peptide associated with
MHC class II Of interest, autocytolytic activity can be detected in most patients
who were treated with CsA plus IFN-
Supported by grants CA15396, CA82583, and AI 24319 from the National Institutes of Health.
Submitted January 9, 2001; accepted March 17, 2001.
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.
Reprints: Yuji Miura, Oncology Center, Johns Hopkins University School of Medicine, Bunting-Blaustein Cancer Research Bldg, Rm 484, 1650 Orleans St, Baltimore, MD 21231; e-mail: ymiura{at}mail.jhmi.edu.
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Top
Abstract
Introduction
, patient 1;
, patient 2; 
, patient 3; 
, patient 4; 
, patient
6; 
, patient 7; 
, patient 8; 
, a patient not administered CsA
and IFN-
