Brain Advance Access originally published online on February 25, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Brain, Vol. 127, No. 5, 996-1008, 2004
© 2004 Guarantors of Brain
doi: 10.1093/brain/awh117
Characteristics of T-cell receptor repertoire and myelin-reactive T cells reconstituted from autologous haematopoietic stem-cell grafts in multiple sclerosis
1 Multiple Sclerosis Research Unit, Department of Neurology and Baylor Multiple Sclerosis Center, 2 Gene and Cell Therapy Center, Department of Medicine, 3 Department of Pathology and 4 Department of Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.
Correspondence to: Dr Jingwu Zhang, Department of Neurology, Baylor College of Medicine, 6501 Fannin Street, NB302, Houston, TX 77030, USAE-mail: jzang{at}bcm.tmc.edu
*These authors contributed equally to this study
 |
Summary |
|---|
 Top Summary IntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Multiple sclerosis is thought to involve aberrant immune responses to myelin autoantigens. Haemato poietic stem-cell transplantation (HSCT) is in clinical trials for progressive multiple sclerosis based on the rationale that it destroys aberrant immune system, while recapitulation of lymphocyte ontogeny might alter the immune system and slow down disease progression. This study was undertaken to analyse characteristics of the T-cell receptor (TCR) repertoire, serum cytokine profile and the T-cell responses to myelin basic protein (MBP) in the reconstituted immune system in progressive multiple sclerosis. The study revealed that, following autologous HSCT, the T-cell immunity recovered in two distinctive phases. The first phase was characterized by limited T-cell immunity as a result of selective expansion of pre-existing T cells commonly expressing the TCR ß chain variable region (TCR BV) 20 and increased serum cytokine production during the first several months. The second phase of T-cell reconstitution coincided with increased thymic T-cell output 9–12 months after HSCT. T cells reconstituted from stem-cell grafts had the distinctive properties of comprehensive T-cell immunity and a broad TCR repertoire. T cells recognizing MBP were initially depleted by immunoablation and rapidly expanded from the reconstituted T-cell repertoire in 12 months. The reconstituted MBP-reactive T cells exhibited a broader epitope recognition repertoire while maintaining the same skewed reactivity pattern compared with that seen at baseline. The findings have important implications in the understanding of the role of HSCT as a potential treatment for multiple sclerosis.
Key Words: haematopoietic stem-cell transplantation; multiple sclerosis; myelin basic protein; T-cell receptor
Abbreviations: CDR3 = complementarity-determining region 3; EDSS = Expanded Disability Status Scale; ELISA = enzyme-linked immunosorbent assay; ELISPOT = enzyme-linked immunospot assay; HSCT = haematopoietic stem-cell transplantation; Ig = immunoglobulin; IL = interleukin; MBP = myelin basic protein; PBS = phosphate-buffered saline; TCR = T-cell receptor; TCR BC = T-cell receptor ß chain constant region; TCR BV = T-cell receptor ß chain variable region; TREC = T-cell receptor rearrangement excision circles
Received November 4, 2003. Revised December 16, 2003. Accepted December 18, 2003.
|
Introduction |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Although the aetiology and pathogenesis of multiple sclerosis remain elusive, there is increasing evidence suggesting that the disease is associated with aberrant immune responses (Martin et al., 1992
; Noseworthy et al., 2000). Among multiple abnormalities that have been reported in multiple sclerosis, aberrant T-cell and antibody reactivity to myelin antigens, such as myelin basic protein (MBP), is thought to play an important role in the disease process (Genain et al., 1990; Ota et al., 1990; Martin et al., 1991; Zhang et al., 1994; Bieganowska et al., 1997; Madsen et al., 1999). These autoreactive T cells have been found to undergo in vivo activation and expansion in the blood and cerebrospinal fluid of patients with multiple sclerosis (Allegretta et al., 1990; Chou et al., 1992; Zhang et al., 1994). There is further evidence suggesting the presence of MBP-reactive T cells in brain lesions of multiple sclerosis patients (Oksenberg et al., 1993; Krogsgaard et al., 2000). In addition to the role of autoreactive T-cell and antibody reactivity, multiple sclerosis is also associated with an abnormal cytokine milieu as part of the inflammatory processes, which is characterized by the increased production of proinflammatory cytokines and chemokines/receptors (Balashov et al., 1999; Zang et al., 2000; Ozenci et al., 2002). At a more general level, aberrant immune responses in multiple sclerosis may be attributable to a dysfunctional immune regulatory network that normally keeps autoreactive T cells and B cells in check (Weber et al., 1998; Mycko et al., 2003). The underlying mechanism is poorly understood at the present time. Currently, the standard treatment options for multiple sclerosis include ß-interferons and glatiramer acetate, whose main mechanism of action is to modulate the immune system (Zhang et al., 2002). Both immunomodulatory agents have been shown to reduce disease activity in relapsing–remitting multiple sclerosis (Noseworthy, 2003). However, these drugs, including mitoxantrone, have a limited clinical effect on the clinical course of secondary progressive multiple sclerosis (European Study Group, 1998; Miller et al., 1999; Hartung et al., 2002). At the present time, control of the disease activity is not satisfactory in a significant proportion of multiple sclerosis patients treated with the drugs, while others do not respond at all. A relatively high percentage of breakthrough disease while on the treatment is another challenge in our effort to treat multiple sclerosis. It is clear that alternative treatment strategies need to be investigated.
Recently, haematopoietic stem-cell transplantation (HSCT) has been tested in clinical trials to treat clinically advanced secondary progressive multiple sclerosis patients (Fassas et al., 1997, 2000, 2002; Burt et al., 1998; Kozak et al., 2000; Openshaw et al., 2000) based on its effectiveness in the treatment of various forms of autoimmune diseases in experimental animals, including experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis (van Bekkum et al., 2002). The commonly used protocol involves high-dose immunosuppression (conditioning regimen) followed by autologous HSCT (Comi et al., 2000). The rationale is that ablation of aberrant immune system followed by reconstitution of the new immune system from haematopoietic stem cells may significantly alter the characteristics of the T-cell responses and other immunological properties, which may improve the clinical course of multiple sclerosis. In more than 100 multiple sclerosis patients treated so far, an autologous HSCT procedure seemed to have some variably positive impact on disease progression of multiple sclerosis (Fassas et al., 1997, 2000, 2002; Burt et al., 1998; Kozak et al., 2000; Openshaw et al., 2000). However, it is unknown whether the observed clinical benefit of autologous HSCT seen in early trials is due to the conditioning regimen and whether the clinical effect of HSCT can be sustained over time. It is speculated that sustained clinical benefit may be achieved if the functional characteristics and the T-cell repertoire of the immune system are significantly altered during reconstitution and such an effect persists over time. This possibility is less likely to occur for autologous HSCT as the new immune system is reconstituted from haematopoietic stem cells of autologous origin. Thus, detailed analysis of the characteristics of the reconstituted immune system, in particular the T-cell receptor (TCR) repertoire and T-cell responses to myelin autoantigens, is important for the understanding of the mechanism of action and for the clinical justification of using autologous HSCT for the treatment of multiple sclerosis, as the procedure is associated with significant mortality (Fassas et al., 1997). The present study was not intended to evaluate the clinical effect of autologous HSCT in a small number of multiple sclerosis patients. It was undertaken to address the above-mentioned specific issues through detailed characterization of various components of the original and reconstituted immune systems. The study, though in a small number of multiple sclerosis patients, has provided important findings that have important implications in the understanding of the role of HSCT in the treatment of multiple sclerosis.
|
Material and methods |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Patients and specimens
Four patients with progressive multiple sclerosis were included in the study. The patients had a mean Expanded Disability Status Scale (EDSS) score of 6.25 and had failed other conventional treatment options. Treatments before the study included ß-interferon, glatiramer acetate, cladribine and mitoxantrone. Blood specimens were collected before the procedures at baseline and at various time-points within the first month then every 3 months after HSCT. CSF was obtained through lumbar puncture at baseline and after HSCT for oligoclonal banding. Neurological examinations and EDSS (Poser, 1983
) were carried out at baseline and every 3 months through the first year after HSCT. MRI examinations of the brain and spine were performed with and without contrast at baseline and 6 and 18 months after HSCT. The study protocol was approved by the institutional human subjects review board.
HSCT treatment protocol
Peripheral blood stem cells were mobilized with granulocyte colony-stimulating factor (10 µg/kg/day) and collected after day 4. Patients received prednisone (1 mg/kg/day) during administration of granulocyte colony-stimulating factor to prevent exacerbation of multiple sclerosis. Cells were selected for CD34+ cells using the Isolex 300i device. Daily leucopheresis was done until 2 million CD34+ cells/kg had been collected. Patients received myeloablative chemotherapy, consisting of cyclophosphamide (90 mg/kg), total body radiation (1000 cGy) and anti-thymocyte globulin (30 mg/kg). A median dose of 3.9 million CD34+ cells/kg (range 2.6–7.4 million) was thawed and infused through a central venous catheter. A median of 1.1 x 104 CD3+ T cells/kg were infused. Granulocyte colony-stimulating factor (5 µg/kg/day) was started on day 1 after transplantation and was given until the neutrophil count reached at least 1000/µl.
Determination of TCR ß chain variable region (TCR BV) gene usage by real-time PCR analysis
Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) and was treated with DNase (RNase-free DNase Set; Qiagen, Hilden Germany) to remove contaminating genomic DNA.
Twenty-five TCR BV and TCR ß chain constant region TCR BC gene segments were cloned by using the TA Cloning kit (Invitrogen, San Diego, CA, USA) and One Shot TOP10 E. coli competent cells (Invitrogen) according to the manufacturer’s protocol. The oligonucleotide sequences of the BV-specific primers are shown in Table 1. cDNA was synthesized from RNA using random primers and Superscript II (Invitrogen) in a 20-µl reaction. TCR BV gene expression was analysed by real-time quantitative PCR. An internal reference control for BV–BC amplification and a non-template control containing no cDNA were added to each reaction. Real-time PCR was performed in 96-well optical PCR plates on an ABI 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Briefly, an aliquot of cDNA sample (0.7 µl) was mixed with 25 pairs of BV-specific primers and one pair of BC primers (0.1 mM in the final solution), respectively, together with SYBR Green PCR Master Mix (Applied Biosystems) to a final reaction volume of 50 µl. All reactions were done in duplicate. The reaction was performed at 50°C for 2 min and at 95° for 10 min as a hot-start activation, which was followed by 40 cycles of reaction at 95°C for 15 s and at 60°C for 1 min. The expression of individual BV genes was calculated based on signal intensity of the PCR reactions according to the following formula:
|
TCR BVn (%) = [2 – (BVn CT – BC CT) x 100) /
(2 – (BV1-25 CT– BC CT) x 100)] x 100 (CT refers to the threshold cycle).
The real-time PCR method used in this study was sensitive and specific for the detection of selective expansion of T cells based on the BV expression pattern. As shown in Fig. 1, selective expansion of BV2 was readily detected by real-time PCR analysis in four peripheral blood mononuclear cell preparations after stimulation with toxic shock syndrome toxin, a superantigen known to activate Vß2+ T cells.
|
Quantitative analysis of TCR rearrangement excision circles by real-time PCR
TCR rearrangement excision circles (TREC) in DNA of PBMCs were performed by quantitative real-time PCR. DNA was extracted from PBMCs using a DNeasy Tissue kit (Qiagen, Valencia, CA, USA) and 100 ng of DNA was added to each reaction with the primers CAC ATC CCT TTC AAC CAT GCT and GCC AGC TGC AGG GTT TAG G (Applied Biosystems). SYBR Green PCR Master Mix (Applied Biosystems) and PCR water were added to a final reaction volume of 50 µl. An additional set of primers to albumin was used to normalize for the relative quantity of genomic TREC (Douek et al., 2000
). The reaction was performed at 50°C for 2 min and at 95° for 10 min as a hot-start activation, which was followed by 40 cycles of reaction at 95°C for 15 s and at 60°C for 1 min. The TREC values were expressed as 2 – 
CT (CT = CT of TREC – CT of albumin).
Immunoscope analysis
PCR reactions were performed with 1 µl of cDNA sample derived from PBMCs in the following amplification mixture: 5 µl 10x PCR buffer (100 mM Tris-HCl, pH 8.3 and 500 mM KCl), 3 µl 25 mM magnesium chloride, 1 µl of 10 mM dNTP mix, 0.5 µl of Taq polymerase (5 U/µl) (Invitrogen, Carlsbad, CA, USA), 20 pmol of primers (specific BV forward primer and BC primer 5'-CGA CCT CGG GTG GGA ACA). The PCR amplification profile used was 30 s at 94°C for denaturation, 30 s at 57°C for annealing and 30 s at 72°C for extension in a total of 40 cycles. Immunoscope analysis was performed with a modified protocol. Two microlitres of BV-specific PCR products were used as templates and run-off reactions were performed with a single internal fluorescent label for each of the 6-FAM (6-carboxy fluorescein)-labelled BC (5',6-FAM-CAC AGC GAC CTC GGG TGG G). The reaction profile consisted of 30 s at 94°C denaturation temperature and 15 cycles at 94°C for 45 s; at 55°C for 45 s, at 72°C for 1 min followed by 72°C for 5 min as an extension step. The resulting PCR products were then denatured in formamide and analysed on an Applied Biosystems 3100 Prism using GeneScan 3.7 software (Perkin-Elmer, Boston, MA, USA). Labelled products were analysed separately as one-colour electrofluorographs. The relative intensity of signal corresponding to complementarity-determining region 3 (CDR3) length was expressed as the area under the experimental peak divided by the area under the control peak found within a Gaussian distribution.
Serum cytokine measurement by enzyme-linked immunosorbent assay (ELISA)
Serum specimens were measured for the concentrations of cytokines using ELISA kits according to the manufacturer’s instructions (Pharmingen, San Diego, CA, USA). Microtitre plates were coated overnight at 4°C with mouse monoclonal antibodies (capturing antibody). Wells were then blocked at 37°C for 2 h with phosphate-buffered saline (PBS) containing 2% bovine serum albumin (Sigma, St Louis, MO, USA) and were subsequently washed four times with 0.02% Tween 20 in a 0.9% NaCl solution. Samples were added in duplicate and incubated for 2 h with a matched biotinylated detecting antibody (0.25 µg/ml of each monoclonal antibody, respectively) in 2% bovine serum albumin/PBS/Tween 20. After washing, avidin-conjugated horseradish peroxidase was added and plates were incubated for 1 h. The substrate used for colour development was 3,3',5,5'-tetramethylbenzidine (Sigma). Optical density was measured at 450 nm using an ELISA reader (Bio-Rad Laboratories, Hercules, CA, USA) and cytokine concentrations were quantitated by Microplate computer software (Bio-Rad) using a double eight-point standard curve. The detection limits for all four cytokines were <15 pg/ml in all assays.
The frequency analysis and epitope recognition of MBP-reactive T cells
Enzyme-linked immunospot (ELISPOT) assays were performed using ELISPOT 
-interferon sets obtained from BD Pharmingen (San Diego, CA, USA) (Pelfrey et al., 2000; Targoni et al., 2001). The ImmunoSpot plates were coated overnight at 4°C with a capture antibody dissolved in PBS. Non-specific binding sites were blocked using 1% bovine serum albumin in PBS for 1 h and washed three times with PBS. PBMC preparations were seeded out at 200 000 per well in triplicate wells in the presence and absence of MBP (40 µg/ml) or the overlapping peptides of MBP (20 µg/ml). RPMI1640 medium containing 5% fetal calf serum was used. Phytohaemagglutinin-protein was used as a positive control. After 48 h, the plates were washed and a biotinylated detection antibody was added and incubated for 12 h at 4°C. Subsequently, streptavidin–horseradish peroxidase in PBS/bovine serum albumin/Tween was added and incubation was performed for 2 h at room temperature. Spots were developed using AEC (Pierce Pharmaceuticals, Rockford, IL, USA) solution for 10–20 min. The reaction was stopped by rinsing plates with water until clear spots were visualized microscopically. The plates/membranes were then air-dried overnight before the plates were subjected to image analysis. The analysis was performed using a Series 1 ImmunoSpot Image Analyzer (Cellular Technology, Cleveland, OH, USA) specifically designed for ELISPOT assay. Digitized images were analysed and results were expressed as number of positive spots per total number of cells per well.
|
Results |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Clinical findings and the general characteristics of the reconstituted immune system after autologous HSCT
Four patients with clinically definite multiple sclerosis were included in the study. Three patients had secondary progressive multiple sclerosis and one had primary progressive multiple sclerosis. Table 2 shows the baseline demographics and histocompatibility leucocyte antigen (HLA) haplotypes of the patients. Mean age at the time of HSCT was 45.5 years, with a mean duration of the progressive phase of multiple sclerosis of 6 years. Patient MS-4 was stable at his 6-month evaluation but declined further follow-up. In the other three patients, the EDSS remained unchanged over the period of 12 months. None of the patients demonstrated any enhancing lesions on follow-up MRI of the brain or spine. MS-3 had several new T2-hyperintense lesions on her follow-up brain MRI at 12 months. No other patient showed any increase in the number of T2 hyperintense lesions on follow-up imaging. CSF was obtained in all patients at baseline and 3 months after HSCT. All samples were abnormal at baseline, showing the presence of oligoclonal bands separate from those present in serum. At follow-up CSF examinations, the number of oligoclonal bands decreased in two patients (MS-2 and MS-3); oligoclonal bands disappeared in MS-4 and persisted in MS-1. None of the patients still being followed has required any further immunosuppressive therapy.
|
Complete immune ablation was achieved in all patients, and was followed by reconstitution of the immune system from mobilized haematopoietic stem cells. As shown in Fig. 2, the white blood cell count reached the pretreatment level
1 month after HSCT and the percentages of monocytes, neutrophils and total lymphocytes remained largely unchanged (Fig. 2A and B). However, although the percentage of CD3+ total T-cell population recovered to the pretreatment level after HSCT, the ratio of CD4+ to CD8+ T cells was altered because of sustained depression of CD4+ T cells (Fig. 2C). Serum levels of immunoglobulin (Ig) G, IgA, IgM and IgE were examined at baseline and every 3 months after HSCT and did not change significantly compared with baseline (data not shown). Furthermore, serum cytokine levels, including interleukin (IL)-12, -interferon, tumour necrosis factor 
, IL-10 and IL-4, fluctuated variably following HSCT and returned to pretreatment low levels at 12 months (Fig. 3). There was significant elevation of serum -interferon, tumour necrosis factor and IL-10 between 3 and 6 months, whereas serum IL-12 production decreased persistently after HSCT in all patients.
|
|
Dynamic changes in the TCR variable gene repertoire and thymic T-cell output
Diversity and comprehensiveness of the T-cell immunity can be reflected in the TCR variable gene repertoire. In a series of real-time quantitative PCR analyses, PBMCs derived from patients at baseline and every 3 months following HSCT were examined for the TCR BV gene distribution pattern using 25 TCR BV-specific primers. The results revealed a highly significant BV skewing for BV20 in all four patients, whereas biased expression of BV1 and BV14 was noted in some but not all multiple sclerosis patients (Fig. 4). The skewed BV gene expression persisted after HSCT for several months before the TCR BV gene distribution returned to a more heterogeneous pattern in 12 months. The overexpression of TCR variable genes (BV20 and BV1) was confirmed in parallel experiments by flow cytometry using conjugated monoclonal antibodies to Vß20 and Vß1. Representative experiments are shown in Fig. 5. Furthermore, PCR-amplified TCR BV transcripts were subject to CDR3 length analysis using immunoscope technique to address whether the overexpression of BV genes represented clonal expansion. As illustrated in Fig. 6, the overexpressed BV20, BV1 and BV14 predominantly exhibited a Gaussian pattern of CDR3 length, suggesting polyclonal distribution. There were a few exceptions that exhibited characteristics of an oligoclonal pattern (e.g. MS3-BV1) (Fig. 6). These findings demonstrated that T-cell populations commonly expressing BV20 and, to a lesser degree, BV14 and BV1, underwent selective expansion and dynamic changes in clonal composition following HSCT, as evident by CDR3 length profiling.
|
|
|
The observed characteristic changes in TCR variable gene repertoire are consistent with selective expansion of pre-existing T cells through a thymus-independent pathway during the first several months (Bomberger et al., 1998
; Douek et al., 2000). The TCR repertoire that was subsequently reconstituted from haematopoietic stem cells had a more heterogeneous distribution pattern that was relatively similar to the pattern at baseline. This is supported by the profile of thymic T-cell output in these patients. In parallel experiments, the thymic output of T cells was measured quantitatively by analysing the numbers of TCREC in peripheral T cells using real-time PCR (Douek et al., 2000). The relative expression of TREC was 27.87 ± 10.97 at baseline and declined drastically to 1.03 ± 1.27 at month 6 after HSCT. The TCREC expression had recovered to 24.76 ± 9.37 by 12 months after HSCT, indicating recovery of thymus-dependent T-cell output within the period between 9 and 12 months.
Precursor frequency and recognition pattern of MBP-reactive T cells recapitulated from autologous haematopoietic stem cells
One of the most important issues to be addressed in this study was related to the recapitulation of autoreactive T cells from autologous haematopoietic stem cells. In this study, MBP was selected for its potential relevance to the pathogenesis of multiple sclerosis. First, we examined the precursor frequency of MBP-reactive T cells using ELISPOT at baseline and every 3 months after HSCT. As shown in Fig. 7, the precursor frequency of MBP-reactive T cells ranged from 2 ± 10–5 to 6 x 10–5 in baseline PBMC specimens of multiple sclerosis patients. The observed frequency of MBP-reactive T cells was higher than that routinely measured in multiple sclerosis patients (Olsson et al., 1992; Hellings et al., 2001). MBP-reactive T cells were depleted by a conditioning regimen and recovered at very low precursor frequency for 9 months after HSCT in all multiple sclerosis patients. However, MBP-reactive T cells reconstituted from autologous haematopoietic stem cells underwent rapid expansion and reached high precursor frequencies that were similar to or exceeded the baseline levels. During that period (from month 9 to month 12), there was no significant change in clinical progression and treatment regimen in these patients.
|
Further analyses were carried out to determine whether the epitope recognition pattern of MBP-reactive T cells occurring 12 months after HSCT was comparable to that of MBP-reactive T cells present at baseline. To this end, PBMC specimens were primed with a set of overlapping 15-mer peptides of MBP and the reactivity to each of the overlapping peptides was measured by ELISPOT based on
-interferon secretion. The results revealed that, at 12 months, the epitope recognition pattern of MBP-reactive T cells was considerably more heterogeneous than that of MBP-reactive T cells present at baseline (Fig. 8). It was noted that the skewed reactivity of MBP-reactive T cells to certain predominant epitopes (e.g. residues 41–59, 81–99, 111–129, 131–149 and 151–170) seen at baseline in these patients persisted at 12 months, though additional epitopes were detected at that time. These experiments concluded that, following sustained suppression after HSCT, MBP-reactive T cells appeared to reconstitute from autologous haematopoietic stem-cell grafts and occurred at frequencies comparable to or higher than pretreatment levels, with a relatively heterogeneous epitope recognition pattern.
|
|
Discussion |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
The study described here has revealed, for the first time, a number of important observations that have direct relevance to the current effort to evaluate the role of HSCT in the treatment of multiple sclerosis. First, the real-time PCR analyses of the TCR variable gene repertoire indicated selective expansion of certain T-cell subsets, as characterized by skewed expression of BV20, BV1 and BV14. Overexpression of BV20 occurred in all individuals. The observed T-cell expansion is likely to have resulted from pre-existing T cells that survived the conditioning regimen. There is evidence indicating that rapid but transient recovery of limited T-cell immunity during the first several months following autologous HSCT is mediated by the expansion of a small population of pre-existing memory T cells—the thymus-independent pathway (Douek et al., 2000
). Consistent with previous reports (Bomberger et al., 1998; Douek et al., 2000) is our finding that the TCR repertoire gradually shifted to a more heterogeneous pattern as new naive T cells were generated from the reconstituted immune system. Although skewed expression of other TCRVB genes has been reported in previous studies unrelated to multiple sclerosis, this is the first description of a common overexpression of BV20 in multiple sclerosis patients after HSCT. The significance of the finding is that the TCR repertoire may be shaped towards selective expansion of BV20+ T cells by the in vivo environment characteristic of multiple sclerosis. For example, a microbial superantigen(s) associated with multiple sclerosis may drive certain T-cell subsets expressing specific BV gene(s) to expand. The CDR3 length analysis of the overexpressed BV transcripts seems to support this possibility.
One of the most important findings described here is related to the occurrence and characteristics of myelin-reactive T cells during reconstitution of the immune system following HSCT. This study shows, for the first time, that MBP-reactive T cells recapitulate from autologous haematopoietic stem cells in multiple sclerosis patients. Following a conditioning regimen, MBP-reactive T cells were initially depleted and recovered subsequently at very low precursor frequencies for several months before they underwent rapid expansion between 9 and 12 months. The finding supports the notion that the T-cell repertoire that initially expanded from pre-existing memory T cells is limited and does not contain sufficient MBP-reactive T cells to mount T-cell responses to MBP, whereas complete reconstitution of a comprehensive T-cell repertoire from autologous haematopoietic stem cells leads to expansion and responsiveness of MBP-reactive T cells. This is further supported by the observation that the thymic output of T cells drastically decreased following HSCT and that it recovered 9–12 months after HSCT, which signals T-cell reconstitution and expansion from stem-cell grafts in the thymus-dependent pathway (Douek et al., 2000). Furthermore, the reconstituted MBP-reactive T cells seem to have a broad epitope recognition repertoire. It is interesting to note that the repertoire of reconstituted MBP-reactive T cells has the imprint of epitope recognition of MBP-reactive T cells present at baseline. That is, they maintained the skewed reactivity towards certain regions of MBP also seen at baseline and broadened the reactivity to additional epitopes of MBP that were not detected before HSCT in these patients. The findings are particularly important as these autoreactive T cells are implicated in the disease processes of multiple sclerosis, and they may also apply to other autoreactive T-cell responses that are potentially involved in multiple sclerosis.
Taken together, the findings described here indicate that, following HSCT, the T-cell component of the immune system seems to recover through two phases, which are characterized by initially limited T-cell immunity as a result of selective T-cell expansion from pre-existing T cells (thymus-independent pathway) and the gradual development and maturation of comprehensive T-cell immunity as new T cells expand from autologous haematopoietic stem cells in a thymus-dependent pathway. The two phases have distinct characteristics in terms of the TCR repertoire, cytokine milieu and autoreactive T cells. Thus, although the immune system is significantly altered during the first phase following HSCT for several months, the functional and structural/phenotypic properties and clonal composition of the reconstituted T cells do not seem to differ significantly from and carry the imprint of the original immune system that was destroyed by autologous HSCT in these multiple sclerosis patients. This is not particularly surprising as haematopoietic stem cells of autologous origin carry all the same genes and have the same lymphocyte ontogeny, and they are not altered per se to develop a different immune system in the same in vivo environment. The findings would indicate that the source of haematopoietic stem cells is important in determining whether the clinical benefit of HSCT will be sustained over time, and would support further investigation to compare immunological properties and the clinical effect of allogeneic versus autologous stem-cell grafts in future studies. It is likely that the immune system reconstituted from a healthy allogeneic source of haematopoietic stem-cell graft may retain some functional and phenotypic characteristics and the cellular composition of the original healthy immune system and may recapitulate the regulatory T-cell component that is critical to sustaining the effect of HSCT.
|
Acknowledgements |
|---|
The work was supported in part by grants from the Richardson Foundation (to J.Z.Z.) and National Institutes of Health (NS41289 to J.Z.Z.). We thank Jeanene De La Rosa and Donna Rill for patient and sample coordination.
|
References |
|---|
|
TopSummaryIntroductionMaterial and methodsResultsDiscussionReferences |
|---|
Allegretta M, Nicklas JA, Sriram S, Albertini RJ. T cells responsive to myelin basic protein in patients with multiple sclerosis. Science 1990; 247: 718–21.
Balashov KE, Rottman JB, Weiner HL, Hancock WW. CCR5+ and CXCR3+ T cells are increased in multiple sclerosis and their ligands MIP-1alpha and IP-10 are expressed in demyelinating brain lesions. Proc Natl Acad Sci USA 1999; 96: 6873–8.
Bieganowska KD, Ausubel LJ, Modabber Y, Slovik E, Messersmith W, Hafler DA. Direct ex vivo analysis of activated, Fas-sensitive autoreactive T cells in human autoimmune disease. J Exp Med 1997; 185: 1585–94.
Bomberger C, Singh-Jairam M, Rodey SMG, Guerriero A, Yeager AM, Fleming WH, Holland HK, et al. Lymphoid reconstitution after autologous PBSC transplantation with FACS-sorted CD34+ hematopoietic progenitors. Blood 1998; 91: 2588–600.
Burt RK, Traynor AE, Cohen B, Karlin KH, Davis FA, Stefoski D, et al. T cell-depleted autologous hematopoietic stem cell transplantation for multiple sclerosis: report on the first three patients. Bone Marrow Transplant 1998; 21: 537–41.[CrossRef][ISI][Medline]
Chou YK, Bourdette DN, Offner H, Whitham R, Wang RY, Hashim GA, et al. Frequency of T cells specific for myelin basic protein and myelin proteolipid protein in blood and cerebrospinal fluid in multiple sclerosis. J Neuroimmunol 1992; 38: 105–34.[CrossRef][ISI][Medline]
Comi G, Kappos L, Clanet M, Ebers G, Fassas A, Fazekas F, et al. Guidelines for autologous blood and marrow stem cell transplantation in multiple sclerosis: a consensus report written on behalf of the European Group for Blood and Marrow Transplantation and the European Charcot Foundation. BMT-multiple sclerosis Study Group. J Neurol 2000; 247: 376–82.[CrossRef][ISI][Medline]
Douek DC, Vescio RA, Betts MR, Brenchley JM, Hill BJ, Zhang L, et al. Assessment of thymic output in adults after haematopoietic stem-cell transplantation and prediction of T-cell reconstitution. Lancet 2000; 355: 1875–81.[CrossRef][ISI][Medline]
European Study Group on interferon beta-1b in secondary progressive multiple sclerosis. Placebo-controlled multicentre randomised trial of interferon-1b in treatment of secondary progressive multiple sclerosis. Lancet 1998; 352: 1491–7.[CrossRef][ISI][Medline]
Fassas A, Anagnostopoulos A, Kazis A, Kapinas K, Sakellari I, Kimiskidis V, et al. Peripheral blood stem cell transplantation in the treatment of progressive multiple sclerosis: first results of a pilot study. Bone Marrow Transplant 1997; 20: 631–8.[CrossRef][ISI][Medline]
Fassas A, Anagnostopoulos A, Kazis A, Kapinas K, Sakellari I, Kimiskidis V, et al. Autologous stem cell transplantation in progressive multiple sclerosis—an interim analysis of efficacy. J Clin Immunol 2000; 20: 24–30.[CrossRef][ISI][Medline]
Fassas A, Passweg JR, Anagnostopoulos A, Kazis A, Kozak T, Havrdova E, et al. Autoimmune Disease Working Party of the EBMT (European Group for Blood and Marrow Transplantation). Hematopoietic stem cell transplantation for multiple sclerosis. A retrospective multicenter study. J Neurol 2002; 249: 1088–97.[CrossRef][ISI][Medline]
Genain CP, Cannella B, Hauser SL, Raine CS. Identification of autoantibodies associated with myelin damage in multiple sclerosis. Nat Med 1990; 5: 170–5.
Hartung HP, Gonsette R, Konig N, Kwiecinski H, Guseo A, Morrissey SP, et al. Mitoxantrone in progressive multiple sclerosis: a placebo-controlled, double-blind, randomised, multicentre trial. Lancet 2002; 360: 2018–25.[CrossRef][ISI][Medline]
Hellings N, Baree M, Verhoeven C, D’hooghe MB, Medaer R, Bernard CC, et al. T-cell reactivity to multiple myelin antigens in multiple sclerosis patients and healthy controls. J Neurosci Res 2001; 63: 290–302.[CrossRef][ISI][Medline]
Kozak T, Havrdova E, Pit’ha J, Gregora E, Pytlik R, Maaloufova J, et al. High-dose immunosuppressive therapy with PBPC support in the treatment of poor risk multiple sclerosis. Bone Marrow Transplant 2000; 25: 525–31.[CrossRef][ISI][Medline]
Krogsgaard M, Wucherpfennig KW, Canella B, Hansen BE, Svejgaard A, Pyrdol J, et al. Visualization of myelin basic protein (MBP) T cell epitopes in multiple sclerosis lesions using a monoclonal antibody specific for the human histocompatibility leukocyte antigen (HLA)-DR2-MBP 85–99 complex. J Exp Med 2000; 191: 1395–412.
Madsen LS, Andersson EC, Jansson L, Krogsgaard M, Andersen CB, Engberg J, et al. A humanized model for multiple sclerosis using HLA-DR2 and a human T-cell receptor. Nature Genet 1999; 23: 343–7.[CrossRef][ISI][Medline]
Martin R, Howell MD, Jaraquemada D, Flerlage M, Richert JR, Brostoff S, et al. A myelin basic protein peptide is recognized by cytotoxic T cells in the context of four HLA-DR types associated with multiple sclerosis. J Exp Med 1991; 173: 19–24.
Martin R, McFarland HF, McFarlin DE. Immunological aspects of demyelinating diseases. Annu Rev Immunol 1992; 10: 153–87.[CrossRef][ISI][Medline]
Miller DH, Molyneux PD, Barker GJ, MacManus DG, Moseley IF, Wagner K. Effect of interferon-beta1b on magnetic resonance imaging outcomes in secondary progressive multiple sclerosis: results of a European multicenter, randomized, double-blind, placebo-controlled trial. European Study Group on Interferon-beta1b in secondary progressive multiple sclerosis. Ann Neurol 1999; 46: 850–9.[CrossRef][ISI][Medline]
Mycko MP, Papoian R, Boschert U, Raine CS, Selmaj KW. cDNA microarray analysis in multiple sclerosis lesions: detection of genes associated with disease activity. Brain 2003; 126: 1048–57.
Noseworthy JH. Management of multiple sclerosis: current trials and future options. Curr Opin Neurol 2003; 16: 289–97.[CrossRef][ISI][Medline]
Noseworthy JH, Lucchinetti C, Rodriguez M, Weinshenker BG. Multiple sclerosis. New Engl J Med 2000; 343: 938–52.
Oksenberg JR, Panzara MA, Begovich A, Mitchell D, Erlich HA, Murray RS, et al. Selection for T-cell receptor V beta-D beta-J beta gene rearrangements with specificity for a myelin basic protein peptide in brain lesions of multiple sclerosis. Nature 1993; 362: 68–70.[CrossRef][Medline]
Olsson T, Sun J, Hillert J, Hojeberg B, Ekre HP, Andersson G, et al. Increased numbers of T cells recognizing multiple myelin basic protein epitopes in multiple sclerosis. Eur J Immunol 1992; 22: 1083–7.[ISI][Medline]
Openshaw H, Lund BT, Kashyap A, Atkinson R, Sniecinski I, Weiner LP, et al. Peripheral blood stem cell transplantation in multiple sclerosis with busulfan and cyclophosphamide conditioning: report of toxicity and immunological monitoring. Biol Blood Marrow Transplant 2000; 6 (5A): 563–75.[CrossRef][ISI][Medline]
Ota K, Matsui M, Milford EL, Mackin GA, Weiner HL, Hafler DA. T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature 1990; 346: 183–7.[CrossRef][Medline]
Ozenci V, Kouwenhoven M, Link H. Cytokines in multiple sclerosis: methodological aspects and pathogenic implications. Mult Scler 2002; 8: 396–404.
Pelfrey CM, Rudick RA, Cotleur AC, Lee J, Tary-Lehmann M, Lehmann PV. Quantification of self-recognition in multiple sclerosis by single-cell analysis of cytokine production. J Immunol 2000 165: 1641–51.
Poser CM, Paty, Scheinberg L, McDonald WI, Davis FA, Ebers GC, et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol 1983; 13: 227–31.[CrossRef][ISI][Medline]
Targoni OS, Baus J, Hofstetter H, Hesse MD, Karulin AY, Boehm BO, et al. Frequencies of neuroantigen-specific t cells in the central nervous system versus the immune periphery during the course of experimental allergic encephalomyelitis. J Immunol 2001; 166: 4757–64.
van Bekkum DW. Experimental basis of hematopoietic stem cell transplantation for treatment of autoimmune diseases. J Leukoc Biol 2002; 72: 609–20.
Weber F, Polak T, Gunther A, Kubuschok B, Janovskaja J, Bitsch A, et al. Synergistic immunomodulatory effects of interferon-beta1b and the phosphodiesterase inhibitor pentoxifylline in patients with relapsing-remitting multiple sclerosis. Ann Neurol 1998; 44: 27–34.[CrossRef][ISI][Medline]
Zang YC, Samanta AK, Halder JB, Hong J, Tejada-Simon MV, Rivera VM, et al. Aberrant T cell migration toward RANTES and MIP-1 alpha in patients with multiple sclerosis. Overexpression of chemokine receptor CCR5. Brain 2000; 123: 1874–82.
Zhang J, Markovic-Plese S, Lacet B, Raus J, Weiner HL, Hafler DA. Increased frequency of interleukin 2-responsive T cells specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. J Exp Med 1994; 179: 973–84.
Zhang J, Hutton G, Zang Y. A comparison of the mechanisms of action of interferon beta and glatiramer acetate in the treatment of multiple sclerosis. Clin Ther 2002; 24: 1998–2021.[CrossRef][ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A M Marmont, F Gualandi, M T van Lint, C Guastoni, and A Bacigalupo Long term complete remission of severe nephrotic syndrome secondary to diffuse global (IV-G) lupus nephritis following autologous, haematopoietic peripheral stem (CD34+) cell transplantation Lupus, January 1, 2006; 15(1): 44 - 46. [Abstract] [PDF]
|
|
|
|
 |
|
 M. M. Herrmann, S. Gaertner, C. Stadelmann, J. van den Brandt, R. Boscke, W. Budach, H. M. Reichardt, and R. Weissert Tolerance induction by bone marrow transplantation in a multiple sclerosis model Blood, September 1, 2005; 106(5): 1875 - 1883. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. A. Muraro, D. C. Douek, A. Packer, K. Chung, F. J. Guenaga, R. Cassiani-Ingoni, C. Campbell, S. Memon, J. W. Nagle, F. T. Hakim, et al. Thymic output generates a new and diverse TCR repertoire after autologous stem cell transplantation in multiple sclerosis patients J. Exp. Med., March 7, 2005; 201(5): 805 - 816. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||