By
From the * Experimental Immunology Branch, National Cancer Institute, National Institutes of
Health, Bethesda, Maryland 20892; Immune Cell Biology Program, Naval Medical Research
Institute, Bethesda, Maryland 20889; § Laboratory of Immunoregulation, National Institute of Allergy
and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892; and ¶ National
Institute on Aging, National Institutes of Health, Bethesda, Maryland 20892
To address the possible role of replicative senescence in human immunodeficiency virus (HIV)
infection, telomere length, telomerase activity, and in vitro replicative capacity were assessed in
peripheral blood T cells from HIV+ and HIV donors. Genetic and age-specific effects on
these parameters were controlled by studying HIV-discordant pairs of monozygotic twins. Telomere terminal restriction fragment (TRF) lengths from CD4+ T cells of HIV+ donors were
significantly greater than those from HIV
twins. In contrast, telomere lengths in CD8+ T cells
from HIV+ donors were shorter than in HIV
donors. The in vitro replicative capacity of
CD4+ cells from HIV+ donors was equivalent to that of HIV
donors in response to stimulation through T cell receptor CD3 and CD28. Little or no telomerase activity was detected in
freshly isolated CD4+ or CD8+ lymphocytes from HIV+ or HIV
donors, but was induced by
in vitro stimulation of both HIV+ and HIV
donor cells. These results suggest that HIV infection is associated with alterations in the population dynamics of both CD4+ and CD8+ T cells,
but fail to provide evidence for clonal exhaustion or replicative senescence as a mechanism underlying the decline in CD4+ T cells of HIV-infected donors.
Aprogressive decrease in numbers of CD4+ T cells is a
prominent feature of HIV infection and AIDS that
correlates with progression of disease and susceptibility to
infection. It appears that excessive destruction of CD4+ T cells
occurs in HIV-1 (HIV) infection, and that an increased rate
of generation of CD4+ cells occurs concurrently, perhaps
reflecting compensatory mechanisms to maintain the population of CD4+ cells (1). CD4+ T cell counts decrease with
progressive HIV infection, indicating that physiologic mechanisms of regeneration are insufficient to compensate adequately for cell loss. It has also been suggested that increased clonal expansion and possible clonal exhaustion of
CD8+ T cells occur in response to HIV infection (2, 3).
Among the factors that can influence replicative potential and clonal expansion, one mechanism that has received
considerable recent attention is the phenomenon of telomere shortening during cell division (4). Observations in
somatic cells indicate that telomere shortening occurs with
each cell division. When telomere shortening has proceeded to some critical minimal length, cell senescence or
arrest of replication is observed, through mechanisms not
yet elucidated. Thus, in the absence of a compensatory mechanism to prevent or reverse replication-associated loss of
telomeric sequences, telomere shortening may contribute to
the finite replicative lifespan of normal somatic cells. A ribonucleoprotein enzyme, telomerase, has the capacity to
add hexanucleotide telomeric repeats to chromosomes, thus
maintaining telomere length (9, 10). Therefore, it was of
interest to determine whether HIV infection results in alterations of telomere length within T cell subpopulations as
a reflection of the replicative history of these cells, and whether the residual replicative potential of T cells is altered in infected individuals.
Isolation and Fractionation of Peripheral Blood T Cells.
Subsets of
peripheral blood T cells were isolated by immunomagnetic beading as previously described (11).
Assay of Telomere Length.
Genomic DNA was isolated from
purified peripheral blood CD4+ and CD8+ T cells, digested with
HinfI and RsaI (Boehringer Mannheim, Mannheim, Germany),
and separated by electrophoresis on a 0.5% agarose gel. Gels were
dried, denatured, hybridized with a 32P end-labeled oligonucleotide (CCCTAA)3 probe, and the gels analyzed by PhosphorImager (Molecular Dynamics, Sunnyvale, CA) as previously reported (11).
PCR-based Telomerase Assay.
The telomerase assay used here
was modified from the telomeric repeats amplification protocol
(TRAP)1 described previously (12). Cell extracts were prepared
in CHAPS lysis buffer, and the telomerase assay and PCR amplification steps were carried out in separate tubes with different
buffers to improve the telomerase efficiency. Telomerase products equivalent to the extracts from 104 cells were used for PCR
with 27 cycles of amplification. The amplified products were separated on a 12% acrylamide gel (NOVEX, San Diego, CA) and
the results analyzed on a PhosphorImager (Molecular Dynamics)
or by exposure to radiographic film. To estimate the activity of
telomerase, serial dilutions of cell extracts were employed, and an
internal standard was included to allow quantitation of PCR by
competition in selected experiments. Controls for the TRAP assay included RNase treatment of cell extracts, single primer (Ts
or Cx alone), and water as template for PCR.
In Vitro Stimulation.
CD4+ T cells were repeatedly stimulated
with immobilized anti-CD3 and anti-CD28 as previously described (11). Population expansion was measured until cell cultures were unresponsive to further stimulation.
To control for known genetic
(13) and age-specific (4) effects on telomere length and
replicative potential, peripheral blood T cells were analyzed
from seven pairs of genetically identical monozygotic twins
who were discordant for HIV infection (Table 1). Total
CD4+ counts ranged from 92 to 562 cells/ml in HIV+ donors, and 500 to 1,516 in their HIV Table 1.
Donor Status
Telomere Length in CD4+ and CD8+ T Cells from HIVinfected and Uninfected Twins.
twins. CD8 counts
ranged from 501 to 1,131 in HIV+ donors, and from 378 to 786 in HIV
donors.
Twin Pair
CD4 count*
%CD45RA/
CD4+
CD8 count*
Age
HIV
diagnosed
Current
clinical status
Antiviral
therapy
HIV+
HIV
HIV+
HIV
HIV+
HIV
yr
1
298
1516
31.7
24.6
1131
786
54
01/89
Pres. PCp (1990)§
AZT, ddI, D4T
2
464
658
27.7
22.5
501
415
31
10/93
Asymptomatic
AZT
3
120
500
14.7
5.9
519
378
44
06/88
Chronic Hepatitis B
AZT, 3TC
4
124
712
42.5
24.7
591
642
30
05/90
Asymptomatic
ddC, 3TC
5
355
1037
51.9
57.3
709
499
29
12/92
Asymptomatic
AZT
6
92
605
51.2
26.9
771
546
54
1989
Asymptomatic
None
7
562
1344
57.7
52.2
1111
359
45
02/90
Asymptomatic
AZT
*
Number of cells/mm3 of peripheral blood.
Percent of CD4+ T cells which are CD5RA positive and CD45RO negative.
§
Presumed Pneumocystis corinii pneumonia noted in 1990, but asymptomatic at the time of apheresis.
If, as has been suggested (14, 15), peripheral blood
CD4+ cells from HIV-positive donors undergo increased
cell division, this difference might be reflected in shortening of telomeres from infected donors when compared with
uninfected twins. Representative telomere terminal restriction fragment (TRF) length analysis of CD4+ and CD8+
T cells from HIV-positive and negative monozygotic twins
is presented in Fig. 1. When comparisons of mean TRF
length were made within each twin pair, it was observed
that TRF length in CD4+ cells on average was significantly
greater in the infected than in the uninfected twins (mean
difference 1.2 ± 0.4 kb) (Fig. 2, A and C). When TRF
length was analyzed in CD8+ populations from HIV-discordant twins, the opposite pattern was observed, with
TRF length in CD8+ cells shorter in HIV-infected twins
than in uninfected twins (mean difference 1.1 ± 0.3 kb)
(Fig. 2, B and C). In HIV-uninfected donors, TRF length
in CD8+ T cells tended to be longer than in CD4+ cells
from the same individual (mean difference 1.2 ± 0.7 kb); whereas in HIV-infected twins, TRF length was greater in
CD4+ cells (mean difference 0.9 ± 0.4 kb).
Telomerase Activity in CD4+ and CD8+ T Cells from HIVinfected and Uninfected Twins.
Two opposing factors are
known to contribute to telomere length: the shortening of
telomeres that occurs with cell division and chromosomal
replication, and the extension of telomeres mediated by
telomerase. To determine whether the TRF length of CD4+
T cells from HIV-infected donors might be influenced by
telomerase activity, telomerase was measured using the
PCR-amplified TRAP assay. Telomerase activity was low
or undetectable in ex vivo isolated CD4+ or CD8+ cells
from a separate panel of 15 (non-twin) HIV-positive donors analyzed or from HIV-negative controls (Fig. 3). Stimulation of CD4+ T cells from HIV-negative donors with antiCD3 and anti-CD28 induced vigorous telomerase activity,
consistent with previous studies (12, 16); and T cells from
HIV-positive donors exhibited similar levels of telomerase
activity after stimulation (Fig. 3).
Replicative Potential of CD4+ T Cells from HIV-infected and Uninfected Twins.
To determine more directly whether
the progressive decrease in CD4+ cell counts during HIV
infection reflects replicative senescence, CD4+ T cells were
stimulated repeatedly with immobilized anti-CD3 and antiCD28 antibodies, and replicative capacity was measured as
the mean number of population doublings (mpd) achieved
before proliferative response ceased (Fig. 4). When CD4+
cells from five sets of discordant twins were compared,
there was no significant difference in the capacity for in
vitro cell division exhibited by HIV-positive and negative
donors (16.6 ± 2.2 mpd for HIV-positive donors and 18.2 ± 2.1 mpd for HIV-negative donors).
The population dynamics of T cell subsets from HIVinfected patients are of considerable interest in efforts to understand the pathogenesis and natural history of AIDS and to design therapeutic intervention. A consistent feature of HIV infection is the progressive decrease in CD4+ T cells that appears to correlate strongly with immune deficiency and susceptibility to opportunistic infection (1). Several lines of experimental evidence have suggested that there is increased destruction of CD4+ T cells in HIV infection, and that there is an increased production rate of CD4+ T cells in HIV-infected individuals, possibly in response to this increased destruction (14, 15). These previous reports suggested that the ultimate decrease in CD4+ T cell levels observed in AIDS might result, at least in part, from exhaustion of the capacity to generate CD4+ cells (17). Since the demonstration 35 years ago that human fibroblasts, a model of normal somatic cells, have a finite capacity for cell division measured in vitro (18), numerous studies have confirmed this observation for cell populations including human T cells. Such findings are consistent with the possibility that a form of clonal exhaustion or senescence might contribute to the immune deficiency seen in AIDS.
The studies reported here have assessed the replicative history and potential of T cells from HIV-infected donors by evaluating several parameters. Telomere length in normal human somatic cells, including T lymphocytes, decreases with cell replication in vitro, on average by 50-200 bp per population doubling (4, 11). A mechanistic explanation for this shortening has been proposed, based on the primer requirement for DNA polymerases and the consequent inability to completely replicate chromosomal termini during cellular S phase. In vivo, telomere length decreases with age of the donor, suggesting that a similar process occurs with aging. These observations have led to a model in which telomere shortening contributes to the finite replicative lifespan of normal somatic cells, with senescence occurring when telomere length reaches some critical minimum that is required for cell replication. Measurement of telomere length has been used to analyze the replicative history of cell populations, an approach that has been applied to analysis of human T cells from healthy uninfected donors. The finding that TRF from CD4+ cells of memory (CD45RO) phenotype are on average 1.4 kb shorter than TRF from naive (CD45RA) CD4+ cells, and that memory cells have less residual replicative potential than naive cells under defined conditions, has been used as the basis for speculation concerning the dynamics of clonal expansion relating these two populations (11).
In the studies reported here, it was found that CD8+ T
cells from infected donors had TRF that were shorter than
those of uninfected donors. Analysis of telomere length has
recently been applied to subsets of human CD8+ T cells.
The CD28 subset of CD8+ T cells from healthy uninfected donors was characterized as a clonally expanded subset and was reported to have TRF that were significantly
shorter than TRF from CD28+CD8+ cells of the same donor (19). Consistent with this finding, it was recently reported that telomere length was reduced in the expanded
CD28
subset of CD8+ cells from HIV-infected patients
(20). The possibility that this difference is related to a functional compromise of CD8+ cells in HIV patients deserves
further attention.
Previous reports have suggested that in HIV-positive donors, increased proliferation of CD4+ T cells may occur in
vivo (14). Were this the case, it might have been expected
that CD4+ cells from HIV-positive twins would, as a correlate of increased clonal expansion, exhibit shorter telomeres than cells from uninfected genetically identical twins.
However, the studies reported here demonstrated that, in
contrast with what was observed for CD8+ cells, CD4+ cells
from HIV-positive donors in fact had TRF significantly longer than those of uninfected twins. Limitations in the
amount of blood that was available from donors in the
present study prevented direct analysis of telomere length
in fractionated CD45RA+ and CD45RO+ subsets of CD4+
cells. However, there was no preferential decrease in the
fraction of CD45RA+ (naive) CD4+ T cells in HIV-infected
twins in the present study, and, therefore, the differences in
telomere length observed between HIV+ and HIV twins
did not correlate with differences in the proportions of these subpopulations. These observations might be interpreted to indicate that, in HIV-infected donors, CD4+ cells
were on average derived from stem cells by a path that involved fewer cell divisions than occur in uninfected individuals, resulting in telomere preservation. For example,
this might occur if destruction of CD4+ cells resulted in regeneration from a pool of stem cells which themselves had
retained longer telomeres. Alternatively, telomere shortening may be uncoupled from cell division in CD4+ cells
from HIV-infected donors, for example, by a mechanism
such as the induction of telomerase. The present study provided no evidence that in vivo telomerase activity contributes to telomere length differences associated with HIV infection, although it cannot be excluded that telomerase
activity in sites other than peripheral blood, or at levels not
detected by the assay employed, plays a role. Finally, the
possibility that HIV infection selectively destroys CD4+ T
cells with shorter telomeres cannot be excluded. These
findings, together with the demonstration that the in vitro
replicative potential of CD4+ cells from AIDS patients was
not diminished relative to that of uninfected twins, suggest
that HIV infection may be associated with alterations in the
population dynamics of both CD4+ and CD8+ T cells, but
that clonal exhaustion or senscence is not a dominant factor
in the progressive loss of CD4+ cells in patients with AIDS.
Address correspondence to Richard J. Hodes, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892.
Received for publication 18 November 1996 and in revised form 23 January 1997.
1Abbreviations used in this paper: mpd, mean population doublings; TRAP, telomeric repeats amplification protocol; TRF, terminal restriction fragment.The authors wish to thank the patients who donated cells for these studies and the physicians who referred those patients to us. Additionally, we thank J. Metcalf and C. Bechtel for facilitating leukopheresis and gathering patient demographics. The authors also wish to thank Dr. R. Walker for his advice and Dr. G. Shearer for his thoughtful comments and review of this manuscript.
This work was supported in part by Army contract DAMD17-93-V-3004, by the Navy Medical Research and Development Command, and by the Henry M. Jackson Foundation. The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Army, Navy, Department of Defense, nor the United States Government.
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