Thymic Export Function and T Cell
Homeostasis in Patients with Relapsing
Remitting Multiple Sclerosis
This information is current as
of June 18, 2017.
Andreas Hug, Mirjam Korporal, Isabella Schröder, Jürgen
Haas, Katharina Glatz, Brigitte Storch-Hagenlocher and
Brigitte Wildemann
J Immunol 2003; 171:432-437; ;
doi: 10.4049/jimmunol.171.1.432
http://www.jimmunol.org/content/171/1/432
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References
The Journal of Immunology
Thymic Export Function and T Cell Homeostasis in Patients
with Relapsing Remitting Multiple Sclerosis1
Andreas Hug, Mirjam Korporal, Isabella Schröder, Jürgen Haas, Katharina Glatz,
Brigitte Storch-Hagenlocher, and Brigitte Wildemann2
M
ultiple sclerosis (MS)3 is a chronic inflammatory disorder of the CNS, and frequently causes considerable
neurologic disability in affected individuals. The
course of disease is mostly relapsing remitting in early stages and
later often becomes secondary progressive with relapse-independent clinical progression. A small proportion of patients experiences a gradual worsening of neurologic function from the onset of
the disease (primary progressive MS). The pathogenic mechanisms
responsible for the damage of neural tissue are unknown. Disease
susceptibility is influenced by the interaction of both genetic and
environmental factors (1, 2), and an immune response targeted
against myelin Ags is generally believed to account for the inflammatory brain and spinal cord lesions. In experimental models of
autoimmune demyelination, a prerequisite for the induction of autoimmunity within the CNS is the activation and extravasation of
circulating autoaggressive T cells with specificity for myelin Ags
(3, 4). Because T cells reactive to components of the myelin sheath
are uniformly detectable in the peripheral T cell repertoire of both
patients with MS and healthy individuals (5, 6), a dysfunction of
Department of Neurology, University of Heidelberg, Heidelberg, Germany
Received for publication November 18, 2002. Accepted for publication April
18, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by Serono Pharma GmbH, Germany and Biogen GmbH,
Germany.
2
Address correspondence and reprint requests to Dr. Brigitte Wildemann, Division of
Molecular Neuroimmunology, Department of Neurology, Im Neuenheimer Feld 400,
D-69120 Heidelberg, Germany. E-mail address: [email protected]
3
Abbreviations used in this paper: MS, multiple sclerosis; DIG, digoxigenin; RRMS,
relapsing remitting MS; RTA, relative enzymatic activity of telomerase; RTE, recent
thymic emigrant; TREC, TCR excision circle; TRF, telomere restriction fragment
length.
Copyright © 2003 by The American Association of Immunologists, Inc.
peripheral immune tolerance mechanisms may contribute to the
immune pathogenesis of MS. One important requirement to keep
circulating autoaggressive T cells under control is the continuous
export of naive T cells from the thymus. The persistent influx of
thymic emigrants regulates T cell homeostasis via the maintenance
of naive T cell diversity (7). Contraction of diversity in the naive
T lymphocyte compartment is associated with the propensity for
the development of autoimmune phenomena in elderly persons and
is a prominent feature of autoimmune dysfunction in rheumatoid
arthritis (8 –10).
In this study, we have assessed the release of naive T cells from
the thymus to test the hypothesis that the continuous thymic output
of naive T lymphocytes and, as a result, T cell homeostasis is
impaired in patients with MS. The thymic T cell production can be
estimated using an intrinsic feature of the TCR rearrangement process that results in the generation of unique TCR excision circles
(TRECs) as a traceable molecular marker in newly produced T
cells (11, 12). As TRECs are stable and do not duplicate during
mitosis (13), they are diluted out in the periphery with Ag-driven
or homeostatic cell division. Therefore, the content of TRECs in
peripheral T cells is a marker of newly synthesized and exported
Ag-naive T cells (recent thymic emigrants (RTEs)) (14, 15). However, TREC levels are not only influenced by the thymus-dependent T cell regeneration process, but also by the degree of proliferation in the peripheral T cell compartment (16). To estimate the
influence of T cell activation and division on the frequencies of
TREC-expressing cells in a chronic inflammatory disease such as
MS, we measured the length of telomeric DNA and telomerase
activity. Telomeres are located at the ends of chromosomes (17,
18) and shorten with each cell division (19, 20). Thus, their length
reflects the proliferative history of peripheral T cells. Telomerase,
an enzyme that is inducible in several cell types, including lymphocytes, may compensate for telomere consumption by adding
telomeric DNA repeats to chromosomal ends (18, 21, 22).
0022-1767/03/$02.00
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Multiple sclerosis (MS) is an inflammatory and possibly autoimmune mediated demyelinating disease of the CNS. Autoimmunity
within the CNS may be triggered by dysfunction of peripheral immune tolerance mechanisms via changes in the homeostatic
composition of peripheral T cells. We have assessed the release of naive T lymphocytes from the thymus in patients with relapsing
remitting MS (RRMS) to identify alterations in the equilibrium of the peripheral T cell compartment. Thymic T cell production
was estimated by measuring TCR excision circles (TRECs) as a traceable molecular marker in recent thymic emigrants. A total
of 46 treatment-naive patients with active RRMS and 49 gender- and age-matched healthy persons were included in the study. The
levels of TREC-expressing CD4ⴙ and CD8ⴙ T lymphocytes were significantly decreased in MS patients, and TREC quantities
overall matched those of 30 years older healthy individuals. The average concentrations of TRECs/106 CD4ⴙ and CD8ⴙ T
lymphocytes derived from MS patients and healthy donors were 26 ⴛ 103/106 and 28 ⴛ 103/106 vs 217 ⴛ 103/106 and 169 ⴛ 103/106,
respectively. To account for any influence of T cell proliferation on TREC levels, we assayed T lymphocytes from additional
patients with MS and normal individuals for telomere length (n ⴝ 20) and telomerase activity (8 MS patients, 16 controls),
respectively. There were no significant differences between CD4ⴙ and CD8ⴙ T cells from MS patients and controls. Altogether,
our findings suggest that an impaired thymic export function and, as a consequence, altered ability to maintain T cell homeostasis
and immune tolerance may play an important pathogenic role in RRMS. The Journal of Immunology, 2003, 170: 432– 437.
The Journal of Immunology
Materials and Methods
Patients
MS patients and controls were recruited at the Department of Neurology,
University of Heidelberg. For TREC analysis in peripheral lymphocytes,
46 individuals (mean age 37 years, range 18 –56 years) with clinically or
laboratory-supported definite MS according to the Poser criteria and 49
gender- and age-matched controls were included in the study (23). All
patients had relapsing remitting MS (RRMS), presented with an acute relapse, and had not yet received treatment with corticosteroids or immunomodulatory agents. The median duration of disease was 2.0 years (average
3.9 years), and patients had previously experienced a median of 2.0 relapses (average 3.1). Seven patients were re-examined within 3 mo (n ⫽ 4),
4 mo (n ⫽ 1), 6 mo (n ⫽ 1), and 7 mo (n ⫽ 1) following resolution of
relapse-associated symptoms. None of these patients had initiated treatment with immunomodulatory agents at second assessment. We additionally included four healthy donors aged between 60 and 80 years. Assessment of lymphocytic telomere length and telomerase activity was
performed in 20 and 8 patients with active RRMS and comparable demographic profiles as well as in 20 and 16 age-matched healthy individuals,
respectively. Patients and controls were informed about the scientific purpose of the study, and all individuals gave written informed consent. The
study was approved by the University of Heidelberg ethics committee.
PBMCs were obtained from 10 ml EDTA blood by density-gradient centrifugation using Ficoll/Hypaque (Biochrom, Berlin, Germany). PBMCs
were separated into CD4⫹ and CD8⫹ populations using magnetic anti-CD4
and anti-CD8 beads (Dynal, Hamburg, Germany), according to the manufacturer’s instructions. In selected experiments, magnetic beads were removed from CD4⫹ T cells using Detachabead CD4 solution (Dynal). This
was followed by positive selection of CD4⫹CD45RA⫹ and
CD4⫹CD45RO⫹ T lymphocytes by the use of CD45RA and CD45RO
microbeads (Miltenyi Biotech, Bergisch-Gladbach, Germany).
Thymocytes were isolated from thymus tissue of a 6-mo-old infant undergoing cardiac surgery. Thymocyte suspensions were obtained by mechanical disruption of thymic tissue in a ground-glass tissue grinder and
subsequent Ficoll density-gradient centrifugation. Cells were washed twice
in serum-free RPMI medium and resuspended in 0.9% NaCl.
Quantification of TRECs by real-time PCR
Total DNA was isolated using DNAzol (Invitrogen, Karlsruhe, Germany),
according to the manufacturer’s instructions. Quantification of TRECs was
performed by SYBR Green real-time quantitative PCR using a GeneAmp
5700 Sequence Detection System (Applied Biosystems, Weiterstadt, Germany). Each amplification was conducted in three independent reactions
using SYBR Green reagents (Applied Biosystems) and reproduced at least
once. Reaction mixtures contained 200 ng total DNA, 3 mM MgCl2, 1 mM
dNTP, 100 nM of each TREC-specific primer (14), 0,75 U AmpliTaq Gold,
and 1 U AmpErase in a total volume of 30 ␮l. Cycling proceeded under the
following conditions: 50°C for 2 min, 94° for 8 min, five cycles of 95° for
1 min, 60°C for 45 s, 72°C for 1 min and 30 s, followed by 35 cycles of
94°C for 45 s, 65°C for 45 s, and 72° for 1 min and 30 s. In each experiment, serial dilutions of a cloned TREC-PCR product were used (104, 103,
102 copies) as a standard for absolute quantification of TREC levels. Results were extrapolated to TREC content per 106 CD4⫹ and CD8⫹ cells or
CD4⫹CD45RA⫹ and CD4⫹CD45RO⫹ cells, respectively.
biotin-labeled primer and synthetic telomeric repeats. The resulting elongation products were amplified by PCR. PCR products were hybridized to
a DIG-labeled telomeric repeat-specific probe bound to a streptavidincoated 96-well plate. The binding reaction was detected with an anti-DIGperoxidase Ab, visualized by a color reaction product, and quantified
photometrically.
Statistical analysis
Statistical analysis was performed using Wilcoxon/Mann-Whitney U test
for unrelated pairs and Pearson’s correlation coefficients. Three age groups
(18 –30 years, n ⫽ 31; 30 – 40 years, n ⫽ 27; and 40 – 60 years, n ⫽ 36)
were independently assessed to account for the age-related decrease of
thymopoiesis. Analysis of covariance was used to assess differences in
CD4⫹ T cell and CD8⫹ T cell TRECs and telomere length between MS
patients and healthy controls, adjusted for age as well as differences between TRECs in naive and memory CD4⫹ T lymphocytes. A p value of
⬍0.05 was considered significant.
Results
TRECs are enriched in phenotypically naive T cells
To illustrate that TRECs identify naive T cells, TREC levels were
determined in naive CD45RA⫹ and memory CD45RO⫹ CD4⫹ T
cells derived from 10 individuals (mean age 31 years, range 21– 44
years). As shown in Fig. 1, TRECs are nearly exclusively detectable in T cells of naive phenotype. TRECs were present at mean
frequencies of 9 ⫻ 103/106 cells (median 5 ⫻ 103/106) in
CD4⫹CD45RA⫹ T lymphocytes as compared with 0.5 ⫻ 103/106
cells (median 0.5 ⫻ 103/106) in the CD4⫹CD45RO⫹ T cell subset.
This is equivalent to an 18-fold enrichment of TRECs in the naive
CD4⫹ T cell subpopulation.
The frequencies of TREC-expressing CD4⫹ and CD8⫹ T cells
are markedly reduced in relapsing MS patients compared with
healthy individuals
The frequencies of TREC-containing T lymphocytes were measured in separated peripheral blood CD4⫹ and CD8⫹ T cells from
46 patients with RRMS in acute relapse and 49 age-matched
healthy persons using quantitative SYBR Green real-time PCR. As
expected, in normal controls, the concentrations of TRECs in both
CD4⫹ and CD8⫹ lymphocyte subpopulations declined exponentially with age (Fig. 2). Between the ages of 18 and 60, the levels
of TRECs dropped by ⬃1 log with a slope of ⫺0.024 TRECs/year
(r2 ⫽ 0.0962) in CD4⫹ T cells and ⫺0.019 TRECs/year (r2 ⫽
0.0993) in CD8⫹ T cells. TRECs were also detectable in lymphocytes of donors aged between 60 and 80 years, although at very
low levels (0.1–7 ⫻ 103 TRECs/106 CD4⫹ lymphocytes and 0.2–
8 ⫻ 103 TRECs/106 CD8⫹ lymphocytes, respectively; data not
shown).
MS patients of any age had significantly lower levels of TRECs
in CD4⫹ and CD8⫹ T cells as compared with normal controls
Telomere length analysis
DNA was extracted from purified T cells using DNAzol (Invitrogen). The
average telomere restriction fragment (TRF) length of CD4⫹ and CD8⫹ T
lymphocyte subsets was determined by the use of the TeloTAGGG Telomere Length Assay (Roche, Basel, Switzerland). A total of 5 ␮g of
genomic DNA was digested with HindI and RsaI, separated on a 0.8%
agarose gel, and transferred onto a nylon membrane. After hybridization
with a digoxigenin (DIG)-labeled telomeric repeat-specific probe and incubation of membranes with a DIG-specific Ab coupled to alkaline phosphatase, the TRFs were visualized by chemiluminescence. The average
TRF length was estimated relative to a m.w. standard that was run in
each gel.
Telomerase activity
Telomerase activity was measured in lymphocytes, thymocytes, and a lymphoma cell line (NC-NC; DSMZ, Braunschweig, Germany) by the use of
the TeloTAGGG telomerase PCR ELISA Plus (Roche) based on the telomeric amplification protocol. Cell extracts were incubated together with a
FIGURE 1. Number of TRECs in naive CD4⫹CD45RA⫹ and memory
CD4⫹CD45RO⫹ T cells from 10 individuals of different ages. TRECs are
nearly exclusively detectable in the naive CD4⫹ T cell subset. Levels of
TRECs are 18-fold enriched in CD4⫹CD45RA⫹ T lymphocytes as compared with CD4⫹CD45RO⫹ T cells.
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Cell separation
433
434
FIGURE 2. Number of TRECs in sorted CD4⫹ (triangles) and CD8⫹
(rectangles) cells from healthy controls of different ages. Best-fit regression
lines are shown. TRECs decline with increasing age, reflecting decreasing
thymopoiesis.
FIGURE 3. A, Number of TRECs in
sorted CD4⫹ cells from healthy controls (triangles) and MS patients (rectangles). Regression lines of controls and MS patients
differ significantly (p ⬍ 0.005). The difference is more pronounced in young MS patients, and the age-dependent slope is markedly reduced (r2 ⫽ 0.0009). B, Number of
TRECs in sorted CD8⫹ cells from healthy
controls (triangles) and MS patients (rectangles). Regression lines of controls and MS
patients differ significantly (p ⬍ 0.005).
FIGURE 4. Relation of TREC numbers in sorted CD4⫹ cells from
healthy controls and MS patients between the age groups 18 –30 years and
the whole study population. The difference of TREC levels is more pronounced in the younger age group (18 –30 years) (factor 14.2) as compared
with the whole study population (factor 8.5).
The yearly rate of decline of TREC-expressing lymphocytes is
demonstrated in Fig. 3. As in controls, the frequencies of TRECcontaining CD8⫹ T cells obtained from subjects with MS decreased as a function of age. Between the ages of 18 and 56, there
was a ⬃1 log drop of TRECs with a slope of ⫺0.019 TRECs/year
(r2 ⫽ 0.0993). In contrast to CD8⫹ T cells, the demise in TRECexpressing CD4⫹ T cells isolated from MS patients was much less
pronounced. In this study, TRECs were detected in nearly constant
quantities in CD4⫹ T lymphocytes throughout all age groups
(slope of ⫺0.003 TRECs/year, r2 ⫽ 0.0009). Overall, the curves
derived from lymphocyte subsets of the MS group were shifted
downward by ⬃1 log as compared with those of the donor group.
Thus, MS patients had levels of TREC-expressing CD4⫹ and
CD8⫹ T lymphocytes equivalent to 30 years older healthy
individuals.
No donor had TREC levels ⬍1 ⫻ 103/106 cells. In contrast, 6
and 7 MS patients (15%), among them individuals in all age
groups, demonstrated levels ⬍1 ⫻ 103 TRECs/106 CD4⫹ T and
CD8⫹ cells, respectively. TREC levels between 1 and 5 ⫻ 103/106
cells were detected in the CD4⫹ and CD8⫹ T lymphocyte subsets
from 2 and 0 donors, respectively, compared with 5 and 9 MS
patients (11%). In contrast, concentrations of TRECs exceeding
⬎100 ⫻ 103/106 cells were present in CD4⫹ and CD8⫹ T cells
from 19 and 14 controls, but from 1 and 3 MS patients only. TREC
levels ⬎500 ⫻ 103/106 cells occurred in 5 (CD4⫹ T cell subset)
and 3 (CD8⫹ T cell subset) controls and in none of the patients.
The frequencies of TREC-expressing CD4⫹ and CD8⫹ T cells
obtained from MS patients are similarly reduced during an
acute relapse and in the relapse-free interval
In seven individuals with MS, concentrations of TRECs in peripheral lymphocyte subsets were repeatedly determined in clinical
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( p ⬍ 0.005 overall and p ⬍ 0.001 in all individual age groups).
The average concentrations of TRECs/106 CD4⫹ lymphocytes in
MS patients and healthy donors were 26 ⫻ 103 (median 17 ⫻ 103)
and 217 ⫻ 103 (median 79 ⫻ 103), respectively (Fig. 3A). CD8⫹
T cells had average values of 28 ⫻ 103 TRECs/106 cells (median
13 ⫻ 103) in subjects with MS and 169 ⫻ 103 TRECs/106 cells
(median 59 ⫻ 103) in controls (Fig. 3B). The most prominent
decrease of TREC-expressing lymphocytes occurred in the CD4⫹
lymphocyte subset isolated from the youngest cohort of MS patients. CD4⫹ T cells of MS patients aged 18 –30 years harbored
TRECs at an average value of 30 ⫻ 103/106 cells (median 18 ⫻
103) compared with an average value of 425 ⫻ 103/106 cells (median 102 ⫻ 103) in healthy donors of the same age. Thus, the
relative reduction of TREC-positive CD4⫹ lymphocytes in this
age group nearly doubled the difference calculated for levels of
TRECs in the CD4⫹ T cell subpopulation of the total study population (Fig. 4). In persons aged 31– 40 years and 41–56 years,
average quantities of TRECs/106 CD4⫹ T cells were 24 ⫻ 103
(median 21 ⫻ 103) and 24 ⫻ 103 (median 13 ⫻ 103) for MS
patients vs 120 ⫻ 103 (median 78 ⫻ 103) and 89 ⫻ 103 (median
72 ⫻ 103) for age-matched healthy subjects, respectively. In the
CD8⫹ T cell compartment, the reduction of TREC-containing
lymphocytes was more evenly distributed among the three individual age groups. CD8⫹ T cells derived from the youngest persons had an average concentration of 45 ⫻ 103 TRECs/106 cells
(median 27 ⫻ 103) in individuals with MS and 270 ⫻ 103 TRECs/
106 cells (median 71 ⫻ 103) in control subjects. In CD8⫹ T lymphocytes isolated from MS patients aged 31– 40 years and 41–56
years, TRECs were detected at an average value of 16 ⫻ 103
(median 7 ⫻ 103) TRECs/106 cells and 24 ⫻ 103 TRECs/106 cells
(median 8 ⫻ 103). This contrasted with average values of 192 ⫻
103 TRECs/106 cells (median 73 ⫻ 103) and 58 ⫻ 103 TRECs/106
cells (median 46 ⫻ 103) in CD8⫹ T lymphocytes obtained from
age-matched controls.
T CELL HOMEOSTASIS IN PATIENTS WITH RRMS
The Journal of Immunology
FIGURE 5. TREC numbers in sorted CD4⫹ (left panel) and CD8⫹
(right panel) cells derived from MS patients (n ⫽ 7) during an acute relapse and in the relapse-free interval. The mean TREC levels did not differ
significantly in the course of disease in either CD4⫹ or CD8⫹ cells, suggesting that the reduced TREC content in MS patients is maintained and
not simply due to an increased proliferation of peripheral T cells during an
acute relapse.
Telomere length in T cell subpopulations does not differ in MS
patients compared with healthy individuals
To investigate whether the reduced TREC concentrations in MS
patients are influenced by an increased peripheral T cell turnover,
we measured the mean telomere length in CD4⫹ and CD8⫹ T cell
subsets from patients with MS (n ⫽ 20) and normal controls (n ⫽
20). As demonstrated in Fig. 6, the mean TRF length declined as
a function of age. There was no significant shortening of telomeres
in lymphocytes derived from MS patients compared with normal
individuals, and in both cohorts there was no difference of TRF
length between CD4⫹ and CD8⫹ T cells. CD4⫹ and CD8⫹ T cells
had a mean TRF length of 10.1 and 9.2 kb (MS patients) and 8.8
and 9.4 kb (controls), respectively.
Telomerase activity in T lymphocytes does not differ in MS
patients compared with healthy donors
Telomere shortening resulting from T cell activation and proliferation may be masked in the presence of a compensatory induction
of telomerase activity. Therefore, we determined the mean relative
enzymatic activity of telomerase (RTA) in T cell subpopulations
from subset patients with MS (n ⫽ 8) and healthy persons (n ⫽
16). Thymocytes and a lymphoma cell line as highly replicating
cells served as a positive control. Telomerase activity was detectable at low levels in peripheral CD4⫹ and CD8⫹ T lymphocytes
from both patients with MS (mean RTA 23 and 6) and normal
donors (mean RTA 27 and 12), respectively, as compared with
thymocytes (mean RTA 232) and lymphoma cells (mean RTA
FIGURE 6. Comparison of mean TRF
length in CD4⫹ (A) and CD8⫹ (B) T cells
from MS patients (n ⫽ 20) (rectangles) and
healthy individuals (n ⫽ 20) (triangles). TRF
length of the two lymphocyte subsets is not
significantly different in MS patients vs
controls.
FIGURE 7. Mean RTA in CD4⫹ and CD8⫹ T cells from MS patients
(n ⫽ 8) and healthy persons (n ⫽ 16). RTA levels in thymocytes and a
neoplastic lymphocyte cell line served as positive controls. There is no
statistically significant difference of lymphocyte RTA between MS patients
and healthy donors.
269) (Fig. 7). CD4⫹ T cells had slightly higher levels of telomerase activity than CD8⫹ T cells, but both CD4⫹ and CD8⫹ T lymphocytes from patients with MS did not express higher telomerase
activity than did the same cell subsets from normal controls.
Discussion
This study demonstrates that T cell homeostasis is substantially
altered in treatment-naive patients with active RRMS. The levels
of circulating TREC-expressing CD4⫹ and CD8⫹ T lymphocytes
were age inappropriately decreased in MS patients with acute relapses, and TREC quantities in lymphocyte subsets derived from
individuals with MS overall matched those of 30 years older
healthy persons. TRECs are a recently defined molecular T cell
marker to measure the frequencies of newly produced T cells exported from the thymus into the peripheral T lymphocyte compartment. They are generated in maturing thymocytes as extrachromosomal circular DNA in the context of the somatic TCR gene
rearrangement (11, 12), and their quantities in peripheral blood
decrease with age as a result of declining thymopoiesis (14, 15).
The correlation of thymic function and circulating RTEs, as assessed by the frequencies of TREC-containing peripheral blood T
cells, is well investigated in animal models, and fractionated removal of single thymic lobes is associated with a corresponding
decrease of RTE numbers in peripheral blood (15, 24). Furthermore, in genetically athymic children with DiGeorge Syndrome
who are unable to produce mature T cells and therefore lack peripheral TRECs, engraftment of thymic tissue is followed by the
appearance and successive increase of TREC-positive lymphocytes in peripheral blood over time (25). Hence, the significant
decline of TREC-expressing circulating T cells in MS patients vs
healthy individuals, as demonstrated in this study, is compatible
with a defective maturation of T cells in MS patients. In concordance with a contracted release of naive T cells from the thymus,
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remission. The frequencies of TREC-expressing CD4⫹ and CD8⫹
T cells were uniformly equivalent as compared with levels detectable during relapses (Fig. 5).
435
436
key players between tolerance and autoimmunity. Such professional regulatory T cells are also present in humans, as shown very
recently independently by several groups (40 – 45). They coexpress
the CD4 and CD25 surface molecules, are anergic to Ag stimulation, and suppress T cell proliferation in a cell-cell contact-dependent manner (40, 41, 43– 45). In addition, they suppress the homeostatic, space-filling (not Ag-driven) T cell proliferation in a
cell culture model (46). Interestingly, although in our study differences in T cell TREC levels were detectable in both the CD4⫹
and CD8⫹ positive subpopulations, the most dramatic decrease of
TREC-containing T cells affected the CD4⫹ T lymphocyte subset
of the youngest MS patients. This was associated with an almost
complete abolition of the age-related decline of the thymic-dependent output of new lymphocytes. This raises the intriguing possibility that the number or function of T lymphocytes with regulatory properties within the CD4⫹ subset may be altered and, like in
murine models, may predispose to organ-specific autoimmunity in
patients with MS.
In conclusion, our study is the first to show that T cell homeostasis is markedly altered in patients with RRMS. Overall, MS patients have levels of TREC-containing CD4⫹ and CD8⫹ T cells
equivalent to 30 years older healthy controls. This abnormality is
detectable in both active phases of the disease and in relapse-free
intervals. Together with the lacking evidence of an accelerated T
cell turnover, as assessed by the length of telomeric DNA repeats
and telomerase activity in circulating T lymphocytes, these findings are compatible with an impaired T cell regeneration in MS.
The altered composition of the peripheral T cell compartment related to this phenomenon may interfere with peripheral immune
tolerance mechanisms that could have impact on future immunomodulatory therapies. In future studies, we will assess whether the
export, function, and deletion of T lymphocytes with regulatory
properties are impaired in MS patients.
Acknowledgments
We thank Brigitte Fritz for excellent technical assistance.
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lymphocyte subsets from MS patients did not show significant
shortening of their telomeres nor increased telomerase activity as
an indirect marker for telomere consumption. This finding suggests
that the dilution of TREC-expressing T lymphocytes as a consequence of an accelerated T cell turnover does not primarily contribute to the marked differences of T cell TREC levels in MS
patients compared with healthy controls. We cannot exclude that
ongoing autoantigen-driven T cell proliferation associated with
acute MS relapses primarily impairs the TREC content of peripheral lymphocytes. However, autoreactive T cells represent a minor
portion within the peripheral T cell repertoire (26, 27), and it is
unlikely that an increased turnover of autoaggressive T cell clones
alone mediates such a pronounced reduction of circulating TRECexpressing T cells in patients with early stage MS. In our study
population, the TREC content was not influenced by the frequency
or severity of prior relapses, and even patients with extremely low
TREC numbers in PBLs (⬍1 ⫻ 103/106 cells) did not differ from
the remaining study population with respect to disease duration,
relapse rate, and disability, as determined by the expanded disability status scale (28). Moreover, in the subset of MS patients that we
assessed serially, the marked reduction of CD4⫹ and CD8⫹ T cell
TREC levels documented in the active phase of the disease was
maintained in the relapse-free interval. These findings suggest that
mechanisms other than immune activation contribute to the lack of
TREC-positive T cells. A substantial reduction of peripheral
TREC levels was also found in patients with rheumatoid arthritis
(29). Unlike MS, this autoimmune disease is characterized by a
high systemic inflammatory activity, which is reflected by an increased T cell turnover, as determined by an age-inappropriate
erosion of telomeric DNA in circulating T cells. Importantly, in
late stages of disease, there is no further shortening of telomeres
compared with healthy controls, indicating that massive peripheral
cell division is not the principal cause for reduced peripheral blood
TREC levels. Furthermore, nonthymectomized patients with myasthenia gravis also have declined frequencies of TREC-expressing T cells (30), suggesting that this phenomenon may coincide
with autoimmune disorders in general. Altogether, our observations indicate that impaired thymic function rather than an accelerated T cell turnover or Ag-driven expansion of autoaggressive T
cell clones is causally related to the pronounced decrease of
TREC-expressing T lymphocytes in patients with RRMS.
The reduced TREC content of circulating T lymphocytes, as
demonstrated in our study, correlates well with previous observations of T cell subset abnormalities in patients with MS and other
autoimmune diseases. Several studies have shown that both NK
cells and phenotypically naive CD4⫹CD45RA⫹ T lymphocytes
are decreased during the active phases of MS (reviewed in Ref.
31). Importantly, the resulting disequilibrium in the composition of
the peripheral T cell compartment may interfere with the maintenance of immune tolerance because a homeostatic shift in the peripheral T cell compartment with up-regulation of phenotypically
naive T lymphocytes and concomitant down-regulation of memory
T cells inhibits the disease activity of MS and experimental allergic encephalomyelitis, as shown for the synthetic immunomodulator linomide (31–33). Furthermore, in murine models, there is
good evidence that athymic animals (e.g., as a result of thymectomy or irradiation of lymphoid tissues) are prone to the development of organ-specific and systemic autoimmune diseases (34).
One important reason for the emergence of autoimmunity in the
experimental setting is the loss of distinct T cell subsets that specifically arise during thymopoiesis and peripheral priming to control and suppress the proliferation of autoaggressive T cell clones
(35–39). The recent characterization of this specialized T cell subset has re-emerged the concept of regulatory T cells as probable
T CELL HOMEOSTASIS IN PATIENTS WITH RRMS
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