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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 242-249
IMMUNOBIOLOGY
T-cell progenitor function during progressive human
immunodeficiency virus-1 infection and after antiretroviral therapy
Dawn R. Clark,
Sjoerd Repping,
Nadine G. Pakker,
Jan M. Prins,
Daan W. Notermans,
Ferdinand W. N. M. Wit,
Peter Reiss,
Sven A. Danner,
Roel A. Coutinho,
Joep M. A. Lange, and
Frank Miedema
From the Department of Clinical Viro-Immunology, Laboratory for
Experimental and Clinical Immunology, CLB, Sanquin Blood Supply
Foundation, the National AIDS Therapy Evaluation Center, the Division
of Infectious Diseases, Tropical Medicine and AIDS, and the Department
of Internal Medicine, Academic Medical Centre, and the Department of
Public Health, Municipal Health Service, Amsterdam, The Netherlands.
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Abstract |
Impairment of T-cell renewal has been proposed as contributing to
CD4+ T-cell depletion in persons infected with human
immunodeficiency virus-1. We analyzed the T-cell development capacity
of progenitors using fetal thymus organ culture. Those who progressed
to AIDS had a dramatic loss in T-cell development capacity shortly
after seroconversion. In contrast, long-term nonprogressors retained progenitor capacity 8 years after seroconversion. Approximately 70% of
patients experienced an improvement in T-cell development capacity
after receiving 6 months of potent antiretroviral therapy. Improvement
in T-cell development in fetal thymus organ culture correlated with an
increase in the number of naive CD4+ T cells in
peripheral blood. Numbers of progenitors in blood and bone marrow after
seroconversion or during therapy did not correlate with the change
observed in T-cell development capacity. These data provide evidence
that HIV-1 infection can interfere with T-cell renewal at the level of
the progenitor cell. Interference with T-cell renewal may contribute to
CD4+ T-cell depletion.
(Blood. 2000;96:242-249)
© 2000 by The American Society of Hematology.
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Introduction |
The effect of human immunodeficiency virus-1 (HIV-1)
infection on T- cell renewal has been studied mainly at the level of peripheral cell proliferation. With the recent evidence that the adult
thymus is a functional organ, interest in the impact of HIV-1 infection
on immune reconstitution from progenitor sources has grown.
There is a considerable body of evidence to support the contention that
HIV-1 infection interferes with hematopoiesis. Defects in stromal
architecture1 and a lack of thymocyte
subsets2,3 have been reported in infected persons.
Moreover, the generation of multiple hematopoietic lineages is blocked
by HIV-1 infection in SCID-hu mice.4,5 CD34+
cells from HIV-1 infected persons display a reduced capacity to develop
cells of erythrocyte, granulocyte, megakaryocyte, and T-lymphocyte
lineages in vitro.6-8 In addition, though CD34+
cells from HIV persons can develop on bone marrow
stroma from infected persons, CD34+ cells from
HIV+ persons do not develop on stroma from uninfected
persons.9 These data suggest that at least some of the
defect lies within the progenitor cells themselves and is not merely a
reflection of the environment in which they reside.
T-cell renewal can be determined by the appearance of new, naive T
cells in the blood. By measuring excision circles resulting from T-cell
receptor gene rearrangement, Koup et al10 estimated the
number of relatively newly developed cells in HIV-1 infected persons.
The number of cells in the blood and lymph nodes expressing excision
circles was lower in HIV+ persons than in control subjects.
They concluded that the output of the thymus is reduced in HIV-1
infected persons. The level of thymic output is likely to be determined
by the amount of functional thymic tissue and the functional capacity
of progenitor cells. Recent data measuring the size of thymic tissue in
HIV+ persons have shown a correlation with numbers of naive
cells in the blood.11
Determination of progenitor function is complicated by the requirement
for ill-defined thymic signals to induce T-cell development from
progenitors. An in vitro T-cell development system, fetal thymus organ
culture (FTOC), has been described in which murine fetal thymus is
repopulated with human CD34+ cells. Most of the literature
describes the development of fetal CD34+ cells in this
system. Studies using adult bone marrow show that adult cells do not
colonize thymus tissue well in the traditional "hanging drop"
method of FTOC.12 In addition, CD34+ cells from
adults appear to develop more rapidly than those from fetuses and the
adult cells lack the sustained double-positive stage that develops when
the cells are grown in fetal thymus.13,14 Another concern is that thymus tissue is traditionally treated with
deoxyguanosine to deplete the resident thymocytes; however, other
thymus cells are also affected by this compound.15 We chose
as a method the direct application of cells to untreated fetal thymus
of T-cell-deficient mice to provide the most normal thymus environment
and to give the adult progenitors the best chance to colonize and develop.
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Patients, materials, and methods |
Subjects
Persons with known dates of seroconversion, who had histories of
consistent sampling and whose pre-seroconversion samples were
cryopreserved, were chosen from the Amsterdam Cohort of Homosexual Men.
Seven long-term non-progressors (LTNP), defined as seropositive for
more than 8 years and with more than 400 CD4+ cells/mL in
the 8th year, whose pre-seroconversion samples were cryopreserved as
part of the Amsterdam Cohort, were selected. Seven age-matched persons
who progressed to AIDS were also selected. Samples from more than 1 year before seroconversion and every 2 years after seroconversion to
the end of follow-up were analyzed. For all progressors and some LTNP,
a 6-month post-seroconversion sample was also analyzed. Two of the
LTNPs and 4 of the progressors were on AZT therapy containing regimens
for the last time point only; for all other samples, the patients were untreated.
For the analysis of anti-retroviral therapy, persons who had
undetectable viral RNA load after 6 months of therapy were assessed. For the Era study (zidovudine or stavudine, lamivudine, nevirapine, indinavir, and abacavir) 6 participants were tested before therapy and
again after 6 and 12 months. In the NUCB2019 trial (zidovudine, lamivudine, and ritonavir), 6 participants were tested before therapy
and again at 6 and 12 months. For the INCAS study (zidovudine, didanosine, and nevirapine), 6 participants were tested before therapy
and again at 6 and 12 months, and a subgroup (n = 3) was tested at 18 months. Four participants in the Era study also donated bone marrow
before the initiation of therapy and after 12 months of therapy.
Samples
All samples were cryopreserved according to a computerized freezing
system that retains the functional capacity of the cells when
they are thawed.16,17 Cryopreserved cells were thawed and
counted by Coulter (Miami, FL) counter, and viability was assessed by
trypan blue dye exclusion. All yields expressed are viable cell yields.
Mice
RAG-1 knockout mice were maintained and bred in microisolator cages
in The Netherlands Cancer Institute (Amsterdam, The Netherlands). Fetal
thymus lobes from 14- and 15-day gestation mice were placed on
Millipore filters (0.45 µm pore size, mixed esters; Bedford, MA)
resting on Netwells (74 µm mesh; Costar, Acton, MA) in 6-well plates.
Organ cultures were grown in Dulbecco's minimum essential medium, 20%
fetal bovine serum (Gibco-BRL, Gaithersburg, MD), 1 µg/mL penicillin,
and 1 U/mL streptomycin and were maintained at 37°C with 5%
CO2.
Progenitor enrichment
Peripheral blood mononuclear cells (PBMC) were isolated from 10 mL
fresh blood or from bone marrow. CD34+ cells were obtained
by positive selection with MACS magnetic beads (Miltenyi Biotech,
Bergisch Gladbach, Germany) according to
manufacturer's instructions. Purity of enriched populations was
greater than 90%, as determined by flow cytometry.
Fetal thymus organ culture
Human cells were placed on each thymus lobe by direct application of
broken cell pellets in multiple 0.2-µL aliquots to the desired
concentration. For unfractionated samples, 5 × 105 to
2 × 106 viable cells per lobe (of 105 samples, 87 were used at a concentration of 7.5 × 105 to 1.3 × 106 cells per lobe) and 1000 to 2200 viable cells
per lobe for progenitor purified samples were used. Eight thymus lobes
were pooled for each determination. Chimeric constructs were maintained
as described above for recipient thymus tissue. After 12 to 14 days in
culture, unless otherwise reported, cells were enzymatically digested
in 0.4 mg/mL collagenase (type F and N, 1:1; Sigma, St. Louis, MO). Cell yield was assessed by a Coulter counter, and viability was assessed by trypan blue exclusion. All culture supernates were tested
for viral replication using p24 enzyme-linked immunosorbent assay
(ELISA), and random checks were made by co-culture with phytohemaglutinin blasts, as previously described.18
FACS analysis
Lymphocyte subsets in peripheral blood were analyzed after
monoclonal antibody staining using the following panel for the NUCB2019
study: CD62L (fluorescein), CD45RA and CD45RO (phycoerythrin), and CD4
and CD8 (PerCP). Naive T cells were those that expressed both CD45RA
and CD62L. For the INCAS and Era studies, the naive determination was
made by expression of CD45RA and CD27. CD62L and CD27 have been shown
to provide similar results for naive cell determination.19
Cells from FTOC were stained for 3-color flow cytometry with monoclonal
antibodies to human CD3 (fluorescein), CD8 (phycoerythrin), and CD4
(Tricolor) or CD45 (Tricolor), major histocompatibility complex (MHC)
class II (fluorescein), and MHC class I (phycoerythrin) as
described.20 All monoclonal antibodies and isotype controls were obtained from Caltag (San Francisco, CA). Cells were run on a
FACScan (Becton Dickinson, Braintree, MA) and analyzed with Cellquest
software (Becton Dickinson). Cells were gated based on low
forward-scatter and side-scatter characteristics for lymphocytes. Gated
cells were analyzed for the expression of CD8/CD4 single-positive and
double-positive subsets and for the expression of CD45 and MHC class
II. Negative gates were set using isotype controls. Organ cultures that
did not display the full spectrum of thymic subsets were excluded from
further analyses.
Total human lymphocytes (THL) were calculated using percentage of cells
expressing CD45 and/or MHC class II and the cell yield. The CD4 and CD8
expression were then calculated from THL. Each plate of every
experiment included 1 organ culture constructed using cells from the
same HIV donor (control). The development of THL of
all plates was corrected to the mean of all the control constructs (ie,
if the control of plate 1 was half the mean of all controls, the THL of
every dish in plate 1 was multiplied by 2). Results are expressed as the number of positive cells/thymus lobe per 10,000 cells in the original inoculum. Statistical analysis was performed using Statview (Abacus, Berkeley, CA), and statistical significance was expressed as a
P value with 95% confidence.
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Results |
FTOC assessment of progenitor function in HIV+ persons
Although unfractionated progenitor sources gave the same result in
FTOC as progenitor purified sources using samples from HIV controls,20 it was possible that
mature cells of HIV+ patients would alter the results
obtained in FTOC. Mature cells from HIV-1-infected patients could
persist or expand in culture and obscure the proper measurement of
progenitor function, or the presence of mature cells could inhibit the
development of progenitors in FTOC. Because insufficient numbers of
purified CD34+ cells could be obtained from cryopreserved
samples for use in FTOC, it was important to determine whether the
development of progenitor cells from HIV+ patients could be
examined, as had been described for HIV participants.
PBMC samples from HIV+ patients were divided into 2 equal
fractions and tested in the chimeric FTOC for their ability to develop along the normal T-cell differentiation pathway. Half was used unfractionated, and the other half was CD34+ cell-enriched
before use. Because the number of CD34+ cells in each
inoculum should have been approximately the same, the relative impact
of mature cells on the development of CD34+ cells could be
assessed. The number of CD4+, CD8+, and
CD4+/CD8+ (DP) cells that developed in FTOC was
slightly higher in the unfractionated group, but this difference was
not statistically significant (Figure 1A).
The CD34 fraction from the progenitor enrichment
yielded few human cells, and these cells did not display thymocyte
phenotypes (not shown).
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| Fig 1.
FTOC assessment of progenitor function in
HIV+ patients.
(A) CD4+, CD8+, and
CD4+/CD8+ (DP) cell development in FTOC from
peripheral blood of HIV+ patients. Samples were split; half
were CD34+ cell enriched ( ), and the other half were
used unfractionated ( ). (B) Kinetics of development in FTOC from
unfractionated PBMC of HIV and HIV+
participants. Immature cells were
CD4+/CD3 (), mature cells ( ) were
CD3+/CD4+ or CD8+ cells ( )
represents double positive cells. Graphs show mean ± SEM of cells
from 3 different donors.
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The kinetics of development was then tested using equal numbers of
unfractionated cells from HIV+ patients and
HIV controls. In both cases, few human
CD3+ cells were present in the thymic construct after 3 days of culture, indicating that mature cells were not retained in the
construct. Immature CD3/CD4+ and
CD8+ (not shown) T cells appeared at approximately 6 days
of culture, and CD4+/CD8+ double-positive cells
could be measured by day 9. Mature CD3+/CD4+ or
CD8+ (not shown) T cells were found by day 12 (Figure 1B).
Although the kinetics of development was the same in both groups,
the number of cells that developed was lower in the HIV+
donor group, particularly in the mature CD3+ subset.
All culture supernates were tested for virus production by p24 ELISA,
and no culture yielded any measurable virus production. The addition of
AZT, saquinavir, or a combination of the 2 drugs did not alter the
reduced development in FTOC from cells of HIV+ patients
(not shown).
HIV-1 infection reduces T-cell development capacity
To assess the effect of HIV-1 infection on T-cell development
capacity during the course of infection, we used peripheral blood
samples, from persons with known seroconversion dates, that had been
cryopreserved as part of the Amsterdam Cohort Studies on AIDS. We
examined the ability of CD34+ cells in these PBMC samples
to develop T cells in FTOC during the course of infection. To determine
whether a lack of T-cell development played a role in CD4+
T-cell depletion, we compared pre-seroconversion and sequential post-seroconversion samples for patients who progressed to AIDS (progressors) and age-matched LTNPs. Progressors and LTNPs had substantially different slopes in CD4+ and CD8+
T cells during the course of infection (Figure
2). Characteristics of those tested are
shown in Table 1.
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| Fig 2.
Profile of peripheral cells during the course of
infection.
Mean ± SEM number of CD4 and CD8 expressing peripheral blood cells
from LTNPs (circles) and progressors (squares) during the course of
infection. For the LTNP group, all patients are represented at all time
points (n = 7). For the progressor group, patients were eliminated from
the study with the AIDS diagnosis; therefore, for time points 0, 0.5, and 2 (n = 7), for time point 4 (n = 6), for time point 6 (n = 5), and for time point 8 (n = 2).
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After seroconversion, progressors showed a substantial reduction in the
pre-seroconversion T-cell development capacity as measured by
CD4+ and CD8+ T-cell development in FTOC
(Figure 3A). Longitudinal analysis showed
that a dramatic loss in T-cell development capacity occurred within 6 months of seroconversion for progressors (Figure 3C). The remaining
T-cell development capacity was depleted during the subsequent years of
infection preceding AIDS diagnosis. In contrast, LTNPs experienced a
slower loss of progenitor development capacity after seroconversion
(Figure 3C); at no time was the difference from pre-seroconversion
statistically significant. After 8 years of infection, LTNPs retained
43.4% of their progenitor capacity compared with their
pre-seroconversion level (Figure 3B). Development in FTOC did not
correlate with the CD4+ T-cell count, the CD8+
T-cell count, plasma HIV RNA load, or T-cell reactivity to anti-CD3 antibody (data not shown). These data strongly support a role for the
loss of progenitor function in CD4+ T-cell depletion and
progression to AIDS.
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| Fig 3.
T-cell development in FTOC from progressors and LTNPs.
(A) CD4 and CD8 expression of cells from FTOC of samples taken during
the course of infection for a representative LTNP (top panels) and
progressor (bottom panels). (B) Shown is the mean (± SEM) number of
CD4+, CD8+, and double-positive cells per lobe
per 10 000 cells in the original inoculum that developed in FTOC.
Pre-seroconversion () and the last post-seroconversion sample tested
(), at AIDS diagnosis for progressors and 8 years
post-seroconversion for LTNPs, are depicted. P values were
calculated with Mann-Whitney U test between the groups and by
Wilcoxon signed rank test within the groups. (C) Longitudinal data for
CD4+ and CD8+ development in FTOC for all
progressors (open squares) and LTNPs (closed circles) combined. Time 0 is the pre-seroconversion sample taken at least 1 year before
seroconversion. P values were calculated with repeated-measures
ANOVA. *P < .001. For the LTNP group, all patients are
represented at all time points (n = 7). For the progressor group,
patients were eliminated from the study with AIDS diagnosis; therefore,
for time points 0, 0.5, and 2 (n = 7), for time point 4 (n = 6), for
time point 6 (n = 5), and for time point 8 (n = 2).
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T-cell development capacity rebounds with anti-retroviral
therapy
CD4+ T-cell counts, including naive CD4+ T
cells, increase after the initiation of potent anti-retroviral
therapy.21-23 If CD4+ T-cell depletion is
partially caused by the inhibition of development, then the increase in
naive CD4+ T-cell numbers after initiation of therapy
should also be related to an improved developmental capacity. To
determine the effect of therapy on T-cell development capacity, we
studied samples from participants in 3 therapy trials: a 3 reverse
transcriptase inhibitor regimen (INCAS),24 a regimen
of 2 reverse transcriptase inhibitors and 1 protease
inhibitor (NUCB2019),25 and a 5-drug regimen including a
protease inhibitor (Era).26 After 6 months of therapy,
approximately 60% of the participants experienced at least a 2-fold
increase in T-cell development capacity (4 of 6 for Era, 4 of 6 for
NUCB2019, 4 of 6 for INCAS) (not shown). T-cell development was
substantially increased after the initiation of therapy in
all 3 groups (Figure 4A). The greatest
change in T-cell development capacity occurred within the first 6 months of therapy, but many participants continued to experience
improvement beyond 6 months. After 18 months of therapy, 10 of the 18 patients tested (3 from Era, 4 from NUCB2019, and 3 from INCAS) had
reached levels of T-cell development capacity comparable to those of
uninfected persons within 18 months of the start of therapy. Most of
the participants (3 from Era, 3 from NUCB2019, and 1 from INCAS)
achieved this level after 6 months of therapy. Of the 11 remaining
participants, 1 INCAS patient and 1 NUCB2019 patient reached normal
levels after 12 months, and another INCAS patient reached them after 18 months (not shown).
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| Fig 4.
Development of T cells in FTOC for participants in
therapy trials.
(A) CD4+ and CD8+ T-cell development before the
start of therapy () and after 6 months of therapy () for all 18 participants. P values were calculated using Wilcoxon
signed-rank test. *P < .001; **P < .005. (B)
Correlation analysis for change in number of naive cells in peripheral
blood and the change in the development of cells in FTOC. Graphs show
the changes in naive CD4+ and CD8+ T cells in
peripheral blood in the first 6 months of therapy compared with the
changes in the development of CD4+ and CD8+
cells in FTOC during the same period for all participants in the
therapy trials. Correlations and P values were determined using
the Spearman rank correlation test.
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We then determined the relationship between an increase in T-cell
development capacity and the number of naive cells circulating in
peripheral blood.23 There was no correlation between
absolute values of these parameters (data not shown). However, the
change in T-cell development capacity between day 0 and 6 months of
therapy showed a positive correlation with the change in the number of circulating naive CD4+ or CD8+ T cells measured
in patients during the same time period (Figure 4B). The strength of
the correlation was lower for CD8 cells, but this might have been
because the development of CD8+ cells in FTOC was less
consistent than of CD4+ cells.
Assessment of bone marrow progenitors
Bone marrow samples were tested from 4 participants in the Era
study. Bone marrow was first enriched for CD34+ cells
before use in FTOC. The number of cells that developed from bone marrow
obtained before therapy was lower than that found for uninfected
persons.13,20 The number of cells that developed in FTOC
was significantly higher after anti-retroviral therapy (Figure
5). Variation of samples was much
lower after treatment, possibly indicating that the effect of HIV on
the bone marrow is mitigated by treatment.
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| Fig 5.
Development in FTOC from bone marrow CD34+
cells.
Mean (± SEM) CD4 and CD8 cell development in FTOC from
CD34+ cells of bone marrow from participants in the Era
trial (n = 4). P values were calculated using the Wilcoxon
signed-rank test. Open bars indicate cell development before therapy;
closed bars, after 6 months' therapy.
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Progenitor cell numbers
It was possible that the observed results could be explained by a
difference in the number of progenitors in the inoculum. Cells from
participants in the seroconverter group and the therapy trial group
were stained for CD34. The number of progenitor cells in the blood
expressing CD34 was lower in LTNPs after seroconversion (Figure
6A). There was no difference in this
parameter before and after seroconversion for the progressor group
(Figure 6A). Participants in the therapy trials had slight decreases in
the number of CD34+ and CD34+/CD7+
cells in peripheral blood after therapy (Figure 6B). In those participants for whom bone marrow was tested, the number of total CD34+ cells increased after therapy, but the number of
CD34+ cells co-expressing CD7 increased only slightly
(Figure 6B).
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| Fig 6.
Number of CD34+ cells.
(A) Number of CD34+ cells in peripheral blood of
progressors and LTNPs (n = 7 for each group) more than 1 year before
(black bars) and 2 years after (white bars) seroconversion. (B) Number
of CD34+ or CD34+/CD7+ cells in
bone marrow (BM) or peripheral blood mononuclear cells (PBMC) of 4 participants in the Era trial before therapy (black bars) and after 6 months of therapy (white bars). P values were determined using
the Wilcoxon signed-rank test.
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Discussion |
The use of the FTOC system for the assessment of T-cell progenitor
capacity from uninfected persons has been previously established. We
have shown that the system can be used to assess the development of
cells from HIV-1 infected persons with a good degree of confidence. The
data obtained from unfractionated and CD34+ cell-enriched
populations were similar to those published for HIV
donors20 in which mature cells contributed little to, or
detracted little from, the development of CD34+ cells in
FTOC. The kinetics of development was the same in the 2 groups, with
cells from HIV+ patients developing far fewer cells
overall. The appearance of double-positive cells, a subset seen only
during T-cell development, in cultures from unfractionated cells
demonstrates that CD34+ cells in the inoculum could enter
the thymus tissue and develop into T cells. Virus production in these
cultures did not appear to play a role in the reduced development
observed with samples from HIV+ persons because the
addition of anti-retroviral drugs to the cultures did not alter the outcome.
Our data provide strong evidence for HIV-1-related decreases in the
T-cell development capacity of progenitors. The difference in the
developmental profiles between progressors and LTNPs suggests that the
HIV-1-related inhibition of T-cell development capacity plays a role
in CD4+ T-cell depletion and progression to AIDS. A
reduction in the developmental capacity of progenitor cells can result
in a decrease in the number of newly produced cells. If cells lost by
natural death or HIV-1-induced killing are not replaced, eventual
depletion of the pool of cells could be expected. This could help
explain the observed impact of HIV-1 on naive cells during infection, though these cells are not considered primary targets for
infection.27 In time, naive cells will be primed to an
activated phenotype, and, if the naive pool is not replenished, the
pool will effectively be depleted.28 These data also
explain the observed loss of naive CD8+ T cells during
infection.28 Impaired progenitor function would be expected
to affect the development of CD4+ and CD8+
cells and to result in a decrease in the number of naive cells of both
phenotypes produced.
Potent anti-retroviral therapy has been reported to result in increases
in the number of CD4+ T cells in the periphery. In the
first few weeks, this increase in CD4+ cells is most likely
the result of a redistribution from the lymphoid tissue because it
concerns mainly cells with a memory phenotype.21-23,29 A
slow increase in the number of naive cells can be observed. Our results
suggest that this increase in naive cells can be related to a
restoration of T-cell development capacity. There was a very strong
correlation between the change in the number of naive CD4+
and CD8+ T cells in the periphery during therapy with the
change in the developmental capacity measured in FTOC for the same
period. Thus, reconstitution of the number of naive circulating
CD4+ T cells is related to an increase in T-cell
development capacity during therapy. Results from FTOC for participants
in therapy trials showed that approximately 70% had a 2-fold or better
increase in T-cell development capacity after 6 months of therapy,
regardless of the regimen. However, only 10 of the 18 persons had
reached normal levels of T-cell development capacity after more than a year of therapy. These data show that the decrease in T-cell
development capacity is, in some cases, reversible but that immune
reconstitution will be slow, as expected from other
studies.30
In this study approximately 30% of participants did not respond with
increases in T-cell development capacity, and changes in the numbers of
naive cells in vivo were extremely small. This may indicate an
irreversible effect of HIV-1 infection on progenitors in some persons.
These persons might also have had previous impairment of progenitor
function, but, without a pre-seroconversion assessment, the actual
cause could not be determined. Additionally, some increases in T-cell
development capacity were not accompanied by increases in naive
CD4+ T cells during therapy. These observations may
indicate potential problems with the other parameter important for
naive T-cell development, the thymus. If the progenitor cell function
increases but the function of the thymus is insufficient, new T cells
will not develop. Indeed, it has recently been reported that only
approximately 30% of infected persons older than 40 had thymi of
substantial size, which correlated with the number of circulating naive
CD4+ T cells.11 This is also consistent with
the report that a fraction of treated patients do not experience immune
reconstitution despite long-term HIV-1 suppression.19 It
remains to be determined which parameters are involved in a patient
experiencing increases in T-cell development during therapy.
The mechanism for HIV-1-related inhibition of T-cell development
capacity has not been determined. One possibility is that the number of
CD34+ cells is reduced in infected persons. There are
conflicting reports on this subject. In our own study, we did not find
a decrease in the number of circulating CD34+ cells after
seroconversion in progressors, but, paradoxically, a slight decrease
was found in LTNPs. We also did not detect an increase in the number of
circulating CD34+ cells in patients on therapy concomitant
with their improved T-cell development in FTOC or increase in naive
cell numbers. In bone marrow, there was an increase in the total number
of CD34+ cells after therapy but only a slight increase in
the mean number of lymphocyte-committed CD7+
cells, with some participants experiencing no increases
at all. It does not appear that the number of progenitors was
responsible for the observations we report.
In this system, a linear relationship between input numbers and output
numbers could be observed between 105 and 106
input cells per lobe, with a log increase in input translating to an
approximately 2-fold increase in output.8 There was
approximately 1 progenitor per 105 cells, and
105 cells can fully reconstitute a fetal thymus lobe.
Therefore, small differences in the number of cells used in this study
would not be responsible for the observed differences in T-cell
development. These data, therefore, indicate that decreased T-cell
development capacity is the result of decreased T-cell development per
T-cell progenitor.
A second possibility is that there is some alteration in the migration
of progenitors such that the CD34+ cells in peripheral blood of
infected persons are not the same as those in uninfected persons. This
is an unlikely explanation because there was no difference in the
numbers of lymphocyte-committed progenitors after seroconversion.
Additionally, development from bone marrow showed the same response to
infection and therapy as peripheral blood.
Numerous other possibilities have yet to be investigated. One is that
the cytokine imbalance seen in HIV-1 infection prevents the
proper development of bone marrow progenitors. Several
pro-inflammatory cytokines, such as TGF- and TNF- , are
produced at inflated levels during HIV-1 infection29,31-35
and are potent inhibitors of hematopoiesis.36-39 During
therapy, the level of inhibitory cytokines has been shown to
decrease,40,41 which may allow progenitors to develop
normally. There are also possible direct effects of virus or viral
products on progenitors38,42-44 that could be reversed with
the decrease in viral replication associated with therapy.
In conclusion, several lines of evidence now point to interference with
T-cell renewal in HIV-1 infection. Our data suggest that part of the
interference is a diminished capacity of progenitors to develop into T
cells. Furthermore, lack of development of new cells would be expected
to contribute to the T-cell depletion that is the hallmark of
HIV-1 infection. With the use of potent antiretroviral therapy, the
function of T-cell progenitors is restored in most patients.
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Acknowledgments |
We thank all who donated blood and bone marrow for this study and
all the clinicians who were involved, particularly R. Kauffmann, N. Foudraine, P. Meenhorst, H. Sprenger, M. van der Ende, and C. ten
Napel. We thank R. van Rij and M. Brower for performing p24 ELISA, the
laboratory of M. T. L. Roos for immunological assays, and E. Tanger for
maintaining the murine breeding colony. Mice were provided by H. Spits
at The Netherlands Cancer Institute.
| |
Footnotes |
Submitted December 16, 1999; accepted February 24, 2000.
Supported by grants from The Netherlands Foundation for
Preventive Medicine, The Netherlands Organization for Scientific
Research, and the Dutch AIDS Foundation. Glaxo-Wellcome sponsored
NUCB2019 and the Era study, Boehringer Ingelheim sponsored INCAS and
the Era study, and Abbott Laboratories sponsored NUCB2019.
Performed as part of the Amsterdam Cohort Studies on AIDS.
Reprints: Frank Miedema, Research and Development Division,
CLB, Sanquin Blood Supply Foundation, Plesmanlaan 125, 1066CX,
Amsterdam, The Netherlands; e-mail: f_miedema{at}clb.nl.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction