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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4464-4471
Reconstitution of the T-Cell Compartment After Bone Marrow
Transplantation: Restoration of the Repertoire by Thymic Emigrants
By
Florence Dumont-Girard,
Etienne Roux,
René A. van Lier,
Geoff Hale,
Claudine Helg,
Bernard Chapuis,
Michel Starobinski, and
Eddy Roosnek
From the Division of Immunology, the Division of Oncology and the
Division of Hematology, the Department of Internal Medicine,
Hôpital Cantonal Universitaire, Geneva, Switzerland; the Lab for
Clinical Viro Immunology, CLB, Sanquin Bloodfoundation and Lab for
Experimental and Clinical Immunology, AMC, Amsterdam The Netherlands;
and the Sir William Dunn School of Pathology, University of Oxford,
Oxford, UK.
 |
ABSTRACT |
We have studied the reconstitution of the T-cell compartment after
bone marrow transplantation (BMT) in five patients who received a
graft-versus-host disease (GVHD) prophylaxis consisting of
methotrexate, cyclosporin, and 10 daily injections (day  4 to day
+5) of Campath-1G. This treatment eliminated virtually all T cells (7 ± 8 T cells/µL at day 14) which facilitated the analysis of the thymus-dependent and independent pathways of T-cell regeneration. During the first 6 months, the peripheral T-cell pool was
repopulated exclusively through expansion of residual T cells with all
CD4+ T cells expressing the CD45RO-memory marker. In two
patients, the expansion was extensive and within 2 months, the total
number of T cells (CD8>>CD4) exceeded 1,000/µL. In the other
three patients, T cells remained low (87 ± 64 T cells/µL at 6 months) and remained below normal values during the 2 years of the
study. In all patients, the first
CD4+CD45RA+RO T cells
appeared after 6 months and accumulated thereafter. In the youngest
patient (age 13), the increase was relatively fast and naive
CD4+ T cells reached normal levels (600 T cells/µL) 1 year later. In the four adult patients (age 25 ± 5), the levels
reached at that time-point were significantly lower (71 ± 50 T
cells/µL). In all patients, the T-cell repertoire that had been very
limited, diversified with the advent of the
CD4+CD45RA+RO T cells. Cell
sorting experiments showed that this could be attributed to the
complexity of the T-cell repertoire of the
CD4+CD45RA+RO T cells that
was comparable to that of a normal individual and that, therefore, it
is likely that these cells are thymic emigrants. We conclude that after
BMT, the thymus is essential for the restoration of the T-cell
repertoire. Because the thymic activity is restored with a lag time of
approximately 6 months, this might explain why, in particular in
recipients of a T-cell-depleted graft, immune recovery is delayed.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
FOR MANY PATIENTS with leukemia,
allogeneic stem cell transplantation is the only curative therapy
available.1-3 Its efficacy is due to the combination of
high-dose chemotherapy, which can be administered despite the
myeloablative effect, the intense transplant conditioning, and the
antileukemic effect of the allogeneic T cells in the graft. After
transplantation, most hematological lineages regain their normal
function rapidly. Unfortunately, this is not the case for the T-cell
lineage and patients can be subject to a variety of infections for
prolonged periods of time.4-9
The reason why T-cell immunity remains impaired, notwithstanding the
fact the peripheral T-cell pool is rapidly repopulated, has been the
subject of a number of studies over the past years. Early reports
already showed the existence of severe imbalances in T-cell
subpopulations.10-12 Furthermore, a high number of T cells
after transplantation appears to be anomalous, expressing numerous
activation or memory markers.13-15 More recently, it has been shown that after the elimination of the peripheral T-cell pool, T
cells can be regenerated through two different
pathways.16-19 One is thymus dependent and might be
considered as a recapitulation of ontogeny. In addition, the T-cell
compartment can be repopulated through peripheral expansion of mature T
cells cotransfused with the bone marrow (BM) graft. The relative
importance of the second pathway is dependent on the activity of the
thymus.16-18,20 Peripheral expansion will only be
significant when the function of the thymus is impaired and its
contribution in young euthymic mice is negligible.
In humans, the same correlation between an insufficient thymic function
and the prevalence of peripheral expansion as a mechanism to restore
the T-cell compartment exists. Therefore, it is likely that in most
patients after BM transplantation (BMT), peripheral expansion is the
primary pathway. Although radiographic imaging of the thymus has shown
that after intense chemotherapy the thymic pathway is
reactivated,18,21 this thymic rebound might become insufficient with increasing age. This has been shown in other studies
that have analyzed the thymus rebound indirectly by measuring the
production of naive CD4+
lymphocytes.15,18,22-25
Whether a unique phenotype exists for naive T cells is still
controversial and, therefore, their identification is not
straightforward. The different splicing forms of CD45 are the surface
markers most widely used to discriminate between naive and memory
cells. Until primed by antigen, naive cells keep the
CD45RA+RO phenotype of thymic emigrants.
Upon activation, T cells become CD45RARO+, but may re-express CD45RA
thereafter. Memory CD8+ T cells may lose the expression of
CD45RO completely,15,26 but this does not seem the case for
CD4+ lymphocytes that remain CD45RARO
double-positive.27 Therefore, the CD4+
lymphocytes with a CD45RA+RO phenotype
that repopulate a patient after eradication of the peripheral T-cell
pool most likely represent thymic emigrants. This is further supported
by the inverse correlation found between the reappearance of
CD4+CD45RA+RO T cells and
age,18 plus the fact that these cells remain absent in the
thymectomized host.28
The contribution of the thymus to the reconstitution of the T-cell
compartment after stem cell transplantation is not precisely known.
Although the production of naive T cells can be taken as representative
for the activity of the thymus, the mere presence of
CD4+CD45RA+RO T cells
cannot. In particular, when the graft contains large quantities of T
cells, the percentage of
CD4+CD45RA+RO T cells might
just reflect the number of naive donor cells that have remained
quiescent. Therefore, thymic activity will be most easily monitored in
recipients of T-cell-depleted stem cell grafts. In this report, we
have studied five patients who received the monoclonal antibody (MoAb)
Campath-1G29,30 in vivo. Besides eliminating cotransfused
donor T cells, the in vivo treatment also lyses residual T cells of
recipient origin so that graft-versus-host disease (GVHD) as well as
graft rejection are avoided. After this therapy, very few mature T
cells were detected in the peripheral blood of the patient, which
allowed a precise analysis of the thymus-dependent and -independent
pathways of T-cell regeneration.
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PATIENTS, MATERIALS, AND METHODS |
Patients.
The clinical characteristics, conditioning regimen, and
posttransplantation immunosuppressive treatments of the five patients are shown in Table 1. All were transplanted
with the marrow of an unrelated donor molecularly matched for HLA-A,
-B, -C, -DR, and DQ . GVHD prophylaxis consisted of Campath-1G (10 mg/d), from day 4 to day +5. Methylprednisolone was administered
on days 4, 3, and 2, at doses of 750, 500, and 250 mg, respectively. From day 1 onward, patients received a 12-hour
infusion of cyclosporin A. The dose was adjusted to attain a trough
level of 200 ng/mL. From around day 30, patients received oral
cyclosporin as indicated in Table 1. Methotrexate was administered at a
dose of 15 mg/m2 (day +1) and 10 mg/m2 (day +3,
+6, +11).
Blood samples and cell sorting.
Peripheral blood cells were collected at various times after
transplantation. Mononuclear cells were obtained after Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. Cells
were fractionated into CD3+CD4+,
CD3+CD8+,
CD4+CD45RA+RO and
CD4+CD45RARO+ cell
populations by fluorescence-activated cell sorting (FACS) (FACSTAR
PLUS, Becton Dickinson, Mountain View, CA) after being stained with
anti-CD3 (mouse-IgG2b-biotinylated) and/or anti-CD4 (mouse-IgG2a or mouse-IgG2b-biotinylated) and/or anti-CD8
(mouse-IgG1) and/or anti-CD45RA-fluorescein isothiocyanate
(FITC) (mouse-IgG1) and/or anti-CD45RO (mouse-IgG2a). Where
needed, the MoAbs were stained with either subclass specific antisera
(Southern Biotechnologies, Birmingham, AL) or streptavidin-red 670 (Life Technologies, Inc, Gaithersburg, MD). Purity of the cell sorted
populations was always more than 97%.
T-cell receptor (TCR)-spectratyping.
Total RNA was purified from 500 to 8,000 T cells using the RNeasy kits
from Qiagen (Qiagen, Hilden, Germany). Two micrograms of
Escherichia coli rRNA was added to each sample as carrier. RNA
was eluted with 45 µL water and lyophilized to reduce the volume down
to 8 µL. First-strand cDNA synthesis was performed with 0.5 mmol/L
dNTPs, 250 ng oligo-dT, and 100 U reverse transcriptase (Life
Technologies, Basel, Switzerland) in a final volume of 15 µL. The
samples were incubated for 30 minutes at 37°C, 30 minutes at
42°C, and denatured for 5 minutes at 95°C. Polymerase chain reaction (PCR) amplification of the cDNA was performed using a radiolabeled constant primer and two primers corresponding to the
variable region of the TCR  -chain, based on the procedure described
by Maslanka et al.31 The following primers were used: BC:5 -AGATCTCTGCTTCTGATGGCT-3, Mix 1-long: BV1,
5-CAGTTCCCTGACTTGCACTC-3, -short: BV5.1,
5-CTCGGCCCTTTATCTTTGCG-3, Mix 2-long: BV12,
5-CAAAGACAGAGGATTTCCTCC-3, -short: BV2,
5-GCTTCTACATCTGCAGTGC-3, Mix 3-long: BV3,
5-GAGAGAAGAAGGAGCGCTTC-3, -short: BV13,
5-GTCGGCTGCTCCCTCCC-3, Mix 4-long: BV6.1,
5-GATCCAGCGCACACAGC-3, -short: BV4,
5-GCAGCATATATCTCTGCAGC-3, Mix 5-long: BV7,
5-CCTGAATGCCCCAACAGC-3, -short: BV8,
5-CCAGCCCTCAGAACCAG-3, Mix 6-long: BV14,
5-GTCTCTCGAAAAGAGAAGAGG-3, -short: BV9,
5-GGAGCTTGGTGACTCTGCTG-3, Mix 7-long: BV20,
5-CACACCCCAGGACCGGCAG-3, -short: BV11,
5-CAGGCCCTCACATACCTCTCA-3, Mix 8-long: BV15,
5-GTCTCTCGACAGGCACAGGC-3, -short: BV17,
5-CCAAAAGAACCCGACAGCTTT-3, Mix 9-long: BV21,
5-GGCTCAAAGGAGTAGACTCC-3, -short: BV16,
5-GAACTGGAGGATTCTGGAGTT-3, Mix 10-long: BV5.3, 5-CCCTAACTATAGCTCTGAGC-3, -short: BV18,
5-GTGCGAGGAGATTCGGCAGC-3, Mix 11-long: BV22,
5-GTTGAAAGGCCTGATGGATC-3, -short: BV24,
5-GGGGACGCAGCCATGTACC-3. The PCR reactions were performed
in 20 µL in presence of: 1 µL cDNA, 50 mmol/L KCl, 10 mmol/L
Tris-HCl pH 8.8, 1 mmol/L MgCl2, 100 µg/mL bovine serum
albumin, 0.2 mmol/L dNTPs, 30 ng BC primer, 3 ng ( -32P)
adenosine triphosphate-labeled BC primer, 30 ng each BV primers, and
0.5 U Taq DNA polymerase (Life Technologies, Basel, Switzerland). Thirty-seven cycles of 30 seconds at 94°C, 30 seconds at 59°C, and 60 seconds at 72°C were followed by a final extension of 5 minutes at 72°C. Three microliters of the PCR samples were mixed with an equal volume of formamide/dye loading buffer, heated at 90°C for 2 minutes and separated on 6% polyacrylamide/urea gels for 4 hours. The gels were dried and exposed with one
intensifying screen for 3 to 12 hours.
| |
RESULTS |
Patients, conditioning, and GVHD prophylaxis.
The characteristics of the five patients enrolled in this study who
received a BM graft from an HLA-A, -B, -C,-DR,-DQ identical donor as a
treatment for chronic or acute leukemia are depicted in Table 1. The
patients received a conditioning regimen based on total body
irradiation and cyclophosphamide. GVHD prophylaxis consisted of
methotrexate, cyclosporin, and the MoAb Campath-1G.29,30 All patients engrafted (polymorphonuclear neutrophils > 0.5 × 109/L), with a median time of 21 days (range,
11 to 30) and remained in complete remission during the time of the
study. Patients 1, 2, and 4 developed acute GVHD stage I-II, which was
successfully treated with systemic steroids. Chronic GVHD occurred in
patient 2 and 3, whereas cytomegalovirus (CMV) viremia was
only observed in patient 1.
Restoration of the T-cell compartment.
Because of the very efficient T-cell depletion by the 10 daily
injections (day 4 to day +5) of Campath-1G, the number of T
cells was very low during the first month after transplantation (7 ± 8 T cells/µL at day 14). Thereafter, two distinct repopulation patterns were observed. In patients 1 and 2, who suffered from GVHD II,
the total number of T cells exceeded 1,000/µL already in the second
month (Fig 1). In patient 2, the vast
majority of the cells were CD8+, which caused the CD4/CD8
ratio to be reversed, a phenomenon that has been observed by
others.10,14,32 In the other three patients with GVHD grade
0-I, expansion was limited. T-cell numbers were approximately 10-fold
lower (87 ± 64 T cells/µL at 6 months) and remained
below normal values during the 2 years of the study.
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| Fig 1.
Time course of the reconstitution of CD4+
and CD8+ T cells in five patients after BMT. ( ), P1;
( ), P2; ( ), P3; ( ), P4; ( ), P5. Shaded area represents
range found in normal controls.
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Limited T-cell repertoire diversity during the first 6 months after
BMT.
During the first months after transplantation, the T-cell compartment
is repopulated mainly through peripheral expansion of mature T cells
that are cotransfused with the graft.19,33,34 Therefore,
T-cell repertoire complexity in recipients of a T-cell-depleted graft
is initially lower than in recipients of an unmanipulated marrow.34,35 To measure the effect of the T-cell depletion with the MoAb Campath-1G, we determined the diversity of the T-cell repertoire at different time points after transplantation. Using the
spectratype method,36 we compared the number of TCRs with a
different CDR3 length of the variable region of the -chain (BV) in
different patients. By testing 21 BVs in two or three samples
containing only low numbers (<104) of T cells, we
obtained results that enabled us to compare the T-cell diversity at
different time points after transplantation. The first panel in
Fig 2 shows the result of three BVs
representative for a polyclonal repertoire of a normal control. It
shows that in a sample of as few as 500 T cells, the number of cells
expressing a BV family with a high frequency such as BV 4 is
sufficiently high to generate seven bands of different CDR3 size.
Because CDR3s of an average size (27 to 33 nucleotides) are much more
abundant than extremely short or long ones,37 the three
most central bands were of higher intensity than the others, indicating
that these bands represented more than one TCR. When a higher number of
T cells was tested (2.103 to 8.103), the
distribution of bands became more Gaussian and with 12 bands
discernible at 8.103 T cells, the heterogeneity of the
CDR3s was almost maximal. BV-families with lower frequencies yielded
different results. For instance, for BV 8, the seven central bands were
present only upon testing 8.103 T cells whereas this was
not yet the case for a BV with a low frequency such as BV 22.
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| Fig 2.
Comparison of spectratypes of a normal control and two
patients 229 and 47 days after transplantation. *The respective lanes
show the bands generated by 500, 2,000, and 8,000 (CD3+)
T cells after PCR amplification with the BV-specific primers.
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This method appeared to be extremely useful to compare the diversity of
the T-cell repertoire in normal individuals with that of patients after
transplantation. Oligoclonal repertoires in patients were readily
discerned by the fact that the number of bands in a BV family was lower
than in the corresponding family of the normal control. More
importantly, a characteristic feature of a restricted repertoire was
that the spectratypes of some BV families were quite similar in
different samples, notwithstanding the fact that they contained higher
numbers of T cells. This is depicted in panels 2 and 3 of Fig 2, which
shows the heterogeneity of BV4/8/22 of two patients after
transplantation. Clearly, the diversity of the T-cell repertoire of
both patients was severely limited. Although the phenomenon was less
pronounced for the BV4 family in patient 1, for the other families
shown, the number of bands generated by 8.103 T cells was
approximately the same as generated by 2.103 cells. This
was particularly striking for BV22 that seemed to consist of very few
clones only. The latter was confirmed by a separate analysis of the
BV22+CD4+ and BV22+CD8+
T cells in patient 1 (Fig 3). Because the
major bands of the BV22 family are found either in the CD8+
or CD4+ FACS-sorted fraction, it is indeed very likely that
these bands represent single clones.
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| Fig 3.
The dominant bands represent single CD4+ or
CD8+ T-cell clones. The data represent the number of
bands generated by either 2,000 or 8,000 cells FACS-sorted on the basis
of the expression of CD3, CD4, or CD8.
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Table 2 shows the analysis of the
repertoire of the CD4+ and CD8+ T cells in all
five patients. To facilitate the comparison of different populations we
expressed the diversity of the respective T-cell repertoires as the
number of BV families of which the seven central bands were detectable.
On the basis of results obtained from normal controls, we estimated
that this was true for BV families of which at least 50 to 100 T cells
in the sample tested used TCRs in a random fashion. The data show that
in the patient samples, very few BV families showed this diversity.
Furthermore, the complexity of the repertoire seemed to reflect the
extent of expansion: TCR diversity was lower in CD8+ T
cells than in CD4+ cells while the repertoire of patients 1 and 2, in whom T-cell expansion had been more extensive, seemed to be
more restricted than that of patients 3 through 5.
The T-cell repertoire is restored by
CD4+CD45RA+thymic emigrants.
After transplantation, mature T cells expand and reconstitute the
peripheral T-cell pool. During this expansion, these cells express a
number of activation markers10,13,14 and acquire the
CD45RO+-phenotype of memory cells. Because not all T cells
expand, the number of T cells that express a
CD45RA+-phenotype, ie, the cells that have remained
quiescent, will be proportional to the number of T cells present before
expansion. Therefore, the percentage of CD45RA+ T cells is
relatively high after peripheral blood stem cell (PBSC) transplantation,38,39 low after BMT,14,39 and
zero when the initial number of T cells has been reduced by T-cell
depletion. During the first 200 days after transplantation, no
CD4+CD45RA+RO T cells were
detected in any of the five patients (Fig
4). Although the number of T cells gradually increased, all T cells
expressed memory markers, indicating that peripheral expansion was
still the only pathway through which the peripheral T-cell pool was repopulated. As a result, no changes in the T-cell repertoire were
observed during this period. Spectratypes remained remarkably constant
with the same bands dominating the respective BV families and without
any noticeable diversification (data not shown).
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| Fig 4.
Time course of the reconstitution of
CD4+CD45RA+RO (RA) T cells
in the two patients with the fast T-cell reconstitution (left) and the
three with the slow reconstitution (right). (), P1; (), P2;
(), P3; (), P4; (), P5. The gray lines/symbols represent the
reconstitution of the CD4+ T cells already shown in Fig
1.
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After approximately 200 days, the first
CD4+CD45RA+RO T cells
appeared. In patient 1 (age 13), these cells rapidly accumulated, while
in the adults (patients 2 through 5, age 25 ± 5), the increase was
much slower. At the same time the T-cell repertoire diversified. The
diversification was due to the high complexity of the T-cell repertoire
of the CD4+CD45RA+RO T cells
(Fig 5). In all patients, T cells sorted on
basis of their CD4+CD45RA+RO
phenotype generated significantly more bands than their
CD4+CD45RARO+ counterparts
(Table 3). Clearly, the diversity of the
T-cell repertoire of the emerging T cells with a naive phenotype was comparable to that of normal controls, while the repertoire of the
CD4+CD45RO+ T cells remained very limited.
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| Fig 5.
The repertoire of the
CD4+CD45RA+RO T cells is
diverse. The respective lanes show the bands generated by 2,000 or
8,000 T cells sorted on the basis of their
CD4+CD45RA+RO or
CD4+CD45RARO+ phenotype.
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Based on these significant differences in T-cell repertoire, we believe
strongly that the
CD4+CD45RA+RO T cells are a
distinct population of thymic emigrants and that, therefore, the role
of the thymus must be considered as essential for the restoration of
the T-cell immunity after BMT.
| |
DISCUSSION |
Malfunctioning of the T-cell compartment causes a significant part of
the life-threatening immunodeficiencies after BMT. Although other
hematopoietic lineages start to function sufficiently already in the
weeks after transplant, T-cell immunity as well as T-cell-dependent B-cell responses might remain impaired for years.5 T-cell
immunity is restored with greater difficulty than other components of
the immune system while the little T-cell memory cotransfused with the
graft is usually not sufficient to substitute the memory of the
recipient that has been destroyed by the chemotherapy and the
transplant conditioning. Indeed, posttransplant T-cell repertoires appear to be of limited diversity,34,36,40-42 even when
large quantities of donor T cells are cotransfused with the
BM.35,43
The distortion of the T-cell repertoire is caused by the fact that
after eradication by chemotherapy, the peripheral T-cell pool will be
initially repopulated through expansion of mature T
cells.19 Because T cells that encounter antigen expand
faster,33,44 a significant part of the final repertoire
might be comprised of limited numbers of dominant T-cell clones
directed against viruses such as CMV,45 against the
histocompatibility antigens of the host,46,47 or possibly
against residual leukemic cells.48,49 As a result, most T
cells express activation markers during the first months after
transplantation,13-15 while the transfused T cells with a
naive phenotype, which have remained quiescent, will be diluted out and
become a minority.
T cells can also be generated from T-cell progenitors through
thymopoiesis. However, in particular in adult recipients of hematopoietic stem cell grafts, the function of the thymus might not be
sufficient to generate a complete new pool of naive T cells. Moreover,
the residual thymic activity might be further reduced by the
combination of irradiation, chemotherapy, and GVHD that causes severe
damage to the thymic microenvironment.50-52
After eradication of the peripheral T-cell pool, restoration of full
immune competence might therefore depend on the capacity of the thymus
to generate a new T-cell repertoire. This has not yet been shown
directly, because monitoring thymus activity in humans is cumbersome.
Recently, several studies have shown that a change in expression of the
different CD45-isoforms on CD4+ T cells can be taken as
evidence of a thymic rebound.17,18,20,24 Because the
CD45RA+RO phenotype of naive T cells is
lost upon activation,53 thymic emigrants are distinct from
the CD45RO+ memory cells generated through peripheral
expansion. Because only CD8+ memory cells may revert to a
CD45RA+RO phenotype,26,54
the increase of
CD4+CD45RA+RO T cells in the
blood of recipients of hematopoietic stem cell grafts must be
proportional to the residual thymic activity of the host. This is
further supported by the correlation between the number of naive
CD4+ T cells and the age of the
recipient18,23,24 and by a case report that shows that
naive CD4+ T cells do not reappear in a recipient who had
been thymectomized before transplantation.28
In this report we have studied the reconstitution of the T-cell
compartment in 5 patients who received the MoAb Campath-1G as
GVHD-prophylaxis. These type of patients are well suited to discriminate the thymus-independent from the thymus-dependent pathway
of T-cell repopulation. Because the treatment eliminates virtually all
T cells, 1 month after transplantation, the number of T cells with a
naive phenotype is negligible when compared with the number of T cells
generated through peripheral expansion. Therefore, any
CD4+CD45RA+RO T cells
detected thereafter must be generated by the thymus, and the
onset of the thymic rebound can be monitored more precisely than in the presence of pretransplant
CD4+CD45RA+RO T cells, which
might expand without acquisition of memory markers.55-57 We
found that during the first 6 months after transplantation, all
peripheral T cells were generated through peripheral expansion. In two
of five patients, the expansion was fast and (supra)normal numbers were
reached within weeks, while in the other three, the number of T cells
increased only slowly. It is noticeable that the two patients with the
rapid increase suffered from GVHD II and that a significant overshoot
of CD8+ T cells was found in the patient with a CMV
infection. The differences in T-cell reconstitution between the
patients were very significant and one could argue that, while these
type of correlations have been suggested by others,58-61
they are most evident when patients are vigorously T-cell depleted.
In all patients,
CD4+CD45RA+RO T cells
started to appear from the sixth month on. This delay of the thymic
rebound is significantly longer than that is observed in patients after
chemotherapy.18 The difference might be explained in
several ways. Firstly, the conditioning received by patients
transplanted with allogeneic stem cells is more intense than standard
chemotherapy. Moreover, the additional immune suppression given after
transplantation might interfere with early intra-thymic T-cell
development.62-64 Secondly, allogeneic stem cell
transplantation could be associated with a slower T-cell
reconstitution61,65 while even mild forms of GVHD can
damage the thymic epithelium.51,52 However, also in a
larger group studied (Roux E, Dumont-Girard F, Helg C, Chapuis B,
Roosnek E; manuscript in preparation), direct correlations between T-cell reconstitution at one hand and immunosuppression or the
occurrence of GVHD on the other were hard to unveil. Because the age of
the patient was by far the most important parameter correlated with
thymic activity, and GVHD and its treatment were in general more severe
in the older patients, these parameters could not be analyzed
separately. CD4+CD45RA+RO T
cells emerged always faster in young patients who received cyclosporin
than in patients over 30 years old who were already off
immunosuppression. Therefore, larger studies in which patients on
immune suppression can be compared with their age-matched controls are
needed to determine to which extent immune suppression hinders the
reconstitution of the T-cell compartment.
The thymic rebound was essential for the restoration of the T-cell
repertoire. Until the appearance of the
CD4+CD45RA+RO T cells,
the repertoire remained extremely limited. By measuring the number of
bands representing different CDR3-lengths of the variable region of the
TCR -chain in very low numbers of T cells, we were able to monitor
the diversification of the repertoire very precisely. We chose the
lowest cell number (2.103) such that in normal controls BV
families with a frequency higher than 5%, ie, the BVs expressed by
more than 100 cells in the sample, generated Gaussian curves of which
the 7 most central bands were always present (Fig 2). In contrast, BV
families with a frequency between 1% and 5% were incomplete, with the
BVs with the lowest frequencies generating one or two bands only. At
the highest cell number (8.103), the seven most central
bands were present in an average of 16 of the 21 BV families (Table 3),
a number that, even when a much higher number (105) of
cells was tested, was never reached in the patient samples before the
advent of the CD4+CD45RA+RO
T cells. The fact that the number of bands generated by as many as
105 T cells in a patient was still lower than that
generated by 8.103 T cells of which the TCR's are randomly
distributed, shows that the diversity in these patients is only a
fraction of that in a normal individual. Therefore, it is not
surprising that their immune reactivity during the first year after
transplantation is severely diminished and that, for the restoration of
the immune response against most antigens, a patient may depend on the
generation of a new T-cell repertoire.
The data shown in this report are characteristic for patients who are
vigorously T-cell depleted and who represent probably the most extreme
example of an immune system with a restricted T-cell repertoire.
However, although TCR diversity might be 2- to 3 logs
higher in recipients of unmanipulated grafts,35 TCR diversity also remains limited in these patients.36,41,43 Although such a repertoire might be sufficient to respond to some antigens, it is not unlikely that the capacity to respond to others is
lost. Therefore, also in these patients, an effective immune response
might depend on the completion of the repertoire by thymic emigrants.
This is suggested by the fact that immunizations to recall antigens are
more efficient in the second year after transplantation, and more
recently, by studies reporting correlations between immune reactivity,
age, and thymic activity.23,25 If so, the administration of
cytokines that could rejuvenate the thymus after transplant might have
a significant therapeutical potential.19,23,66,67
| |
FOOTNOTES |
Submitted May 13, 1998;
accepted July 26, 1998.
F.D.-G. and E.R. contributed equally to this work.
Supported by a grant of the Swiss National Science Foundation
(32-043622.95).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Eddy Roosnek, PhD, Unité
d'Immunologie de Transplantation, Hôpital Cantonal Universitaire
de Genève, 24 rue Micheli-du-Crest, CH-1211 Genève 14, Switzerland; e-mail: edro{at}hcuge.ch.
| |
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Abstract
Introduction