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Prepublished online as a Blood First Edition Paper on October 10, 2002; DOI 10.1182/blood-2002-06-1644.
PLENARY PAPER
From the Institut für Virologie und
Immunbiologie, Julius-Maximilians-Universität, Würzburg,
Germany; Deutsches Primatenzentrum, Göttingen,
Germany; Innere Medizin II, Universität des
Saarlandes, Homburg/Saar, Germany; and Institut für
Pathologie, Julius-Maximilians-Universität, Würzburg,
Germany.
HIV infection leads to reduced numbers and increased turnover of
CD4+ T cells in blood. However, blood represents only 2%
of the total lymphocyte pool, and information about other organs is
lacking, leading to controversy about the effects of HIV infection on
T-cell homeostasis. Therefore, we have determined phenotype and
turnover of lymphocyte subsets in various tissues of macaques.
Infection with simian immunodeficiency virus (SIV) resulted in
increased proliferation rates of T cells in all organs. Despite reduced CD4 counts in blood, absolute numbers of CD4+ T cells were
increased in spleen and lymph nodes and remained stable in nonlymphoid
organs such as liver, lung, bone marrow, and brain during the
asymptomatic phase, indicative for an altered tissue distribution. In
animals killed with first signs of AIDS, total body CD4 counts and
proliferation rates had returned to control levels, whereas thymocytes
were almost completely absent. Our data show that a drastically
increased turnover in the early stages of HIV infection, driven by a
generalized immune activation rather than a homeostatic response to
CD4+ T-cell destruction, is followed by exhaustion of the
regenerative capacity of the immune system.
(Blood. 2003;101:1213-1219) Determining the mechanisms leading to
CD4+ T-cell loss in human immunodeficiency virus
(HIV)-infected patients is crucial for the understanding of the
pathogenesis of AIDS.1 The common picture holds that
T-helper cells decline steadily although there may be differences in
the rate between various organs.2-7 The observation of a
rapid rebound of CD4+ cell numbers in blood after
initiation of highly active antiretroviral therapy
(HAART)8,9 led to speculation that the infection results
in increased peripheral T-cell proliferation. This increased turnover
was thought to be a homeostatic response to a massive destruction of
T-helper cells, which cannot be compensated for in the long run by the
regenerative capacities of the immune system. Subsequently, direct
evidence for an increased fractional proliferation rate of blood
CD4+ T cells has accumulated,10-18 whereas the
effect of HIV infection on total T-cell turnover is still being
discussed.19-21 Therefore, other groups postulate
decreased production and peripheral turnover as the cause of T-helper
cell depletion.13,22 The discrepancies between the various
theories are due mainly to a lack of information about the situation in
other parts of the body.
So far, the effects of HIV infection on T-cell pool and T-cell turnover
have been investigated mostly in blood.10-12,17 However, blood lymphocytes represent only 2% of the total lymphocyte pool, and
their composition differs from that within the
organs.23,24 From other organs, only information about
changes in the proportions of CD4+ T cells and fractional
proliferation rates is available.3-5,13,18,25 Absolute
figures for the entire body have been extrapolated solely from
blood-derived data.8-10,13,15 However, because there is evidence that HIV infection also alters the distribution of lymphocytes between different organs,26 it is necessary to directly
determine the absolute numbers of lymphocyte subsets for the various
compartments.24 For obvious reasons, this can be achieved
only in an animal model. Therefore, we have determined absolute numbers
of lymphocyte subsets in 14 different tissues of uninfected and simian
immunodeficiency virus (SIV)-infected macaques.
Animals and viruses
Preparation of lymphocytes from different organs
Flow cytometric analysis Isolated cells from the various organs (about 2 × 105) or citrated blood samples were subjected to 3-color flow cytometry as described recently.29 The following antibodies were used: antimonkey CD3 FN18 (M. Jonker, TNO (Nederlandse Organisatie voor toegepast-natuurwetenschappelijk onderzoek), Rijswijk, The Netherlands) biotinylated using standard techniques, CD20-fluorescein isothiocyanate (FITC) (B1, Coulter, Krefeld, Germany), CD4-phycoerythrin (PE) (OKT4, Ortho, Neckargemünd, Germany; L200, Pharmingen, Heidelberg, Germany), CD8-FITC (RPAT8, Pharmingen), CD29-FITC (4B4, Coulter), and Ki-67-PE (Ki-67, Dako, Hamburg, Germany). After lysing of erythrocytes and fixing the cells with fluorescence-activated cell sorter (FACS) lysing solution (Becton Dickinson, Heidelberg, Germany), bound biotinylated antibodies were detected with streptavidin-coupled CyChrome (Pharmingen, Hamburg, Germany). Cells were analyzed on a FACScan flow cytometer (Becton Dickinson). Quadrants were set according to the staining pattern obtained with isotype-matched control antibodies except for the bimodal distribution of CD29, where markers were set between the 2 CD29low- and CD29high-expressing populations. For determination of absolute numbers of lymphocyte subsets, the proportion of the respective lymphocyte subset within a forward and side light scatter gate including CD3+ T cells, CD20+ B cells, and CD3 CD8+ natural killer (NK) cells was
multiplied by the absolute number of lymphocytes obtained with a
Coulter counter (blood) or by counting in a Neubauer chamber as
described above (organs). Because fewer than 5% of macaque and human T
cells in blood and LNs express neither CD4 nor CD8, the
respective T-cell subsets negative for 1 of the 2 antigens consist
mainly of the reciprocal population. Thus, because the expression of
Ki-67 was measured only in combination with CD8, the expression of
Ki-67 determined for CD8 T cells will be referred to as
expression on CD4+ T cells. Some CD4+ T cells
also express the CD8 homodimer. Because we did not perform double
staining for both CD4 and CD8 in one tube (except for thymus), this
small subset of fewer than 5% of T cells added not only to the
CD4+ but also to the CD8+ T cells. This might
have resulted in a slight overestimation of the CD8+ T-cell
compartment. To estimate the contribution of the lymphocytes in
the organs investigated to the total lymphocyte pool, peripheral blood
lymphocytes (PBLs) were isolated from 45 mL blood of 3 uninfected animals 1 day prior to necropsy. After counting and labeling
with carboxyfluorescein diacetate succinimidyl ester (CFSE), these cells were reperfused into the same animals. The proportion of CFSE-positive cells among cells isolated from different organs 20 hours
later was determined by flow cytometry, and the absolute number of
labeled cells was calculated.
Statistical analysis All data are expressed as mean ± SEM. The proportion and absolute numbers of lymphocyte subsets from uninfected and SIV-infected animals was compared using the Mann-Whitney U test. Correlation between different parameters was assessed using the Spearman rank correlation coefficient. The significance level was set at P .05.
In this study, we have included a total of 43 juvenile rhesus monkeys. Eleven uninfected monkeys with a mean weight of 3821 ± 175 g were used as controls. Of the 32 SIV-infected monkeys, 12 (mean weight, 4138 ± 222 g) had to be humanely killed due to signs of AIDS (Table 1) between 14 and 67 weeks after infection (wpi) (mean, 38 ± 6 wpi). Twenty animals (mean weight, 4453 ± 173 g) were killed at predetermined time points between 12 and 78 wpi (mean, 38 ± 5 wpi) without signs of AIDS. CD4+ cell counts in blood were decreased from 932 ± 72 cells per microliter in uninfected animals to 584 ± 71 in asymptomatic monkeys and to 503 ± 137 in animals with AIDS. Cell-associated viral load increased from 606 ± 221 infectious cells per 106 PBMCs in asymptomatic monkeys to 3349 ± 1452 in monkeys with AIDS. We have first determined the absolute numbers of lymphocytes in several
organs (Figure 1). In uninfected monkeys,
the spleen represented the largest lymphoid organ. The sum of 6 lymph
node (LN) regions investigated resulted in a similar number of
lymphocytes. As nonlymphoid organs, we assessed liver, lung, BM, and
the brain (1.8 × 106 lymphocytes). The absolute numbers
obtained for all organs except lung, where we have isolated fewer
cells, are comparable to those found in humans23 and other
animal species. From these organs a total of 1.2 × 1010
lymphocytes was calculated, which, according to previous estimates for
humans,23 represented at least 50% of the total
lymphocyte pool. Age, weight, and sex did not correlate with total
lymphocyte numbers. To determine the total yield with a different
method, we counted ex vivo carboxyfluorescein diacetate succinimidyl
ester (CFSE)-labeled peripheral blood lymphocytes in the different
organs 20 hours after reperfusion. CFSE-positive cells accounted for
0.1% to 0.3% of lymphocytes in the various organs. Taken together,
more than 50% of the reperfused cells were found again in all 3 animals. The remaining cells may have been lost during purification but
more likely reside within compartments not investigated with this
technique (for example, gut and other LN regions). Because we have
assessed more than 50% of the total lymphocyte pool in both lymphoid
and nonlymphoid compartments, our results can be viewed as
representative for the whole body. Additionally, we have quantified
lamina propria lymphocytes (LPLs) in 5 uninfected animals, which added
up to approximately
5.1 × 108 ± 2.2 × 108 lymphocytes at
an estimated total gut weight of 200 g. Other organs, such as
heart (about 2 × 106 lymphocytes), kidney (about
2 × 107), submandibular gland (about
1 × 106), and tonsils
(3.7 × 107 ± 6.6 × 106),
which we have investigated in some animals, did not contribute significantly to the total number of lymphocytes.
After infection, the absolute numbers of lymphocytes increased during the asymptomatic phase in all organs except blood, resulting in a 3-fold overall increase in the sum of the organs investigated (Figure 1). In animals with AIDS, absolute numbers of lymphocytes further increased in most nonlymphoid organs (liver, lung, brain) and declined in LNs, spleen, and bone marrow compared with asymptomatic animals but remained increased compared with uninfected monkeys. In blood, the lymphocyte counts were reduced in animals with AIDS compared with preinfection levels. The most dramatic changes in lymphocyte numbers were observed in thymus. In 8 of 11 animals with AIDS, thymocyte numbers were reduced by more than one order of magnitude. The normal composition of lymphocytes varies between different organs.
Accordingly, the CD4/CD8 ratios in uninfected animals ranged from 2.5 in tonsils to less than 0.5 in liver, bone marrow, brain, and LPLs
(Figure 2A). For the sum of all major
compartments investigated, the mean CD4/CD8 ratio was 1.01 ± 0.03.
The CD4/CD8 ratios declined after infection, as expected, in all organs
investigated (Figure 2B). However, this decrease differed between the
organs, ranging from only 25% in thymus (of mature
CD3high-expressing CD4 or CD8 single-positive
thymocytes) to more than 70% in nonlymphoid organs such as the lung
and the gut of asymptomatic animals (Figure 2B). In animals with AIDS,
the CD4/CD8 ratios further decreased in most organs. Two of these
monkeys (nos. 1962, 9340) showed exceptionally high CD4/CD8 ratios, a
fact that has been often reported for monkeys with a rapid progressor
phenotype,30 resulting in a large variation of the CD4/CD8
ratio of animals with AIDS.
Despite the reduced proportions of CD4+ T cells in all
organs of asymptomatic animals, the absolute numbers of
CD4+ T cells were decreased only in blood (Figure
3). Moreover, in thymus, lymph nodes, and
spleen, the absolute numbers of CD4+ T cells were increased
compared with uninfected monkeys. Even in nonlymphoid organs, which
exhibited the most dramatic decrease in the proportion of
CD4+ T cells, this population did not decline in absolute
numbers (Figure 3). In the sum of all organs investigated,
CD4+ T cells increased significantly from
3.5 × 109 ± 3.9 × 108 in uninfected
monkeys to 6.6 × 109 ± 6.3 × 108 cells
in the asymptomatic phase (P
Absolute numbers of CD8+ T cells were significantly
increased in all organs except blood (Figure 3), resulting in a total
3-fold increase from
3.5 × 109 ± 3.1 × 108 in uninfected
animals to 1.15 × 1010 ± 1.4 × 109 in
asymptomatic monkeys (P Both augmented replenishment of the peripheral T-cell pool from thymic and extrathymic sources or increased peripheral proliferation could explain the observed increase in total T-cell numbers. Unfortunately, thymic output is not accessible to direct measurement. Methods to detect recent thymic emigrants by T-cell receptor excision circles (TRECs) may not properly reflect thymic function, because peripheral proliferation has a greater influence on TREC levels than thymic output.31 Our observation of increased numbers of mature thymocytes is consistent with previous findings of increased levels of cell proliferation in the thymus of SIV-infected macaques32 and abundant thymic tissue in HIV patients33 and shows that thymic function is preserved, if not increased, during the asymptomatic phase of HIV and SIV infection. As to the proliferation of peripheral T cells, we determined the
expression of Ki-67, a nuclear antigen expressed from G1 to M phase, in
cells from blood, inguinal LNs, spleen, liver, lung, and BM of 10 uninfected, 16 asymptomatic, and 10 diseased monkeys. The other LN
regions and organs were investigated in fewer animals. Thymus was not
included in the analysis because expression of Ki-67 was limited mainly
to CD3
The average fractional proliferation rate for all organs increased from
4.48% ± 0.39% to 9.14% ± 0.97% for CD4+ T cells
and from 3.92% ± 0.43% to 12.98% ± 1.36% for CD8+
T cells. This fact, combined with the increased absolute counts of the
2 T-cell subsets, led to a 4-fold increase of the total number of
Ki-67-expressing CD4+ T cells from
1.5 × 108 ± 1.5 × 107 in uninfected
monkeys to 5.9 × 108 ± 8.7 × 107 in
asymptomatic monkeys (P
CD4+ T cells can be functionally and phenotypically
delineated into naive and memory/effector subsets. We have used the
expression level of CD29 to differentiate between these 2 subsets.
CD29high-expressing memory cells show higher proliferation
rates than CD29low-expressing naive cells29
and are preferentially infected by HIV and SIV.34,35 The
proportion of memory cells differed between the various organs from
less than 20% in LNs to more than 50% in nonlymphoid organs such as
liver and lung (Figure 6).
In asymptomatic animals, the percentage of memory cells remained
essentially unchanged, whereas it was reduced among CD4+ T
cells in animals with AIDS. These results show that the presumably higher depletion of memory cells by the infection is balanced during
the asymptomatic stage either by proliferation within this subset or
conversion of naive cells into this cell type. Later in course of the
infection, the increasing drain on memory CD4 cells could not
be compensated, because proliferation rates were reduced, resulting in
a strong decrease in absolute numbers of memory cells in 4 of 5 animals
with AIDS (Figure 7).
To determine the effect of viral replication on T-cell turnover, we
stratified the animals according to the cell-associated viral load
(Figure 8). The fractional proliferation
rate of CD4+ T cells was increased only in animals with low
to medium viral load. In animals with high viral load, the
proliferation rate was comparable to uninfected animals. Ki-67
expression of CD8+ T cells increased with virus titers and
reached a maximum in monkeys with a medium viral load. In animals with
high viral load, the proliferation rate of this subset decreased again
but was still higher than in uninfected macaques. According to these
findings, it seems that 2 forces are acting in opposite directions.
Increased viral replication leads to increased immune activation and
proliferation but also results in increased death of CD4+ T
cells. At high viral load, a low to absent T-helper cell response might
also influence the proliferative capacity of CD8+ T cells.
In uninfected individuals, the production rate (thymic output plus peripheral proliferation) and death rate of T cells is balanced, and the net increment of the total T-cell pool is zero. Given the increased number of thymocytes and the increased proliferation rate of peripheral T cells, the production rate was increased in our cohort of animals. Because the absolute numbers of CD4+ and CD8+ T cells were increased, the production rate must be higher than the death rate. We have used the difference in the absolute cell numbers between uninfected and asymptomatic animals to determine the net increment rate. According to the formula it = Ninf/Nuninf, where i is the increment rate, t the duration of infection, and N represents the total number of cells of an individual infected animal and the mean of all uninfected animals, respectively, we calculated a mean increment rate of 0.0024 ± 0.0007 per day for CD4+ T cells and 0.0055 ± 0.0010 for CD8+ T cells in asymptomatic monkeys. These numbers are by far lower than the infection-induced increases in the proliferation rates as measured by Ki-67 expression (%Ki-67+ cells in infected minus %Ki-67+ cells in uninfected animals: 0.0466 and 0.0906 for CD4+ and CD8+ T cells, respectively). Therefore, the death rate must be increased by the infection for both subsets as well, assuming that the span of time of Ki-67 expression during cell cycle and the thymic output is not drastically changed.
In this work, we determined for the first time absolute numbers of several lymphocyte subsets in solid organs of SIV-infected macaques. Because we have sampled about 50% of the total lymphocyte pool,23 our data provide a solid base for estimates of immunodeficiency virus-induced changes in total T-cell pool and turnover. Our results confirm previous reports of increased fractional proliferation rates of both CD4+ and CD8+ T-cell subsets after HIV and SIV infections in blood and LNs8-13,15,17,18 and extend this observation to other organs. Moreover, we could show that the absolute proliferation rate is increased as well and that the pattern of distribution between different organs is altered. Although circumstantial evidence of an altered distribution of CD4+ T cells between the different organs existed previously,15,24,26 our findings provide the first quantitative description of this effect. The most prominent result of our study, however, is an expanded size of the total CD4+ T-cell pool upon SIV infection. This was completely unexpected in the light of decreased absolute numbers in blood and reduced proportions in all organs. Enlargement of LNs and spleen is frequently found in asymptomatic HIV patients.36,37 In addition, it has been reported that CD4+ cell numbers in tonsils were not decreased in asymptomatic patients.6 These facts, however, have not been taken into account for calculations of the absolute numbers of lymphocytes.13,25 Several hypotheses have been put forward to explain the reduced numbers of CD4+ T cells in blood during HIV and SIV infection. In the low turnover model, it has been postulated that the infection interferes with the replenishment of CD4+ T cells.19,22 However, similar to our results, most studies have found increased fractional proliferation rates of CD4+ T cells in blood of HIV-infected patients and SIV-infected macaques.10,11,14,15 In some studies, low CD4 counts in blood outweighed increased proliferation rates, resulting in a reduced total turnover for this T-cell subset,12,13,25 interpreted as evidence for the low turnover model. Our finding of an increased proliferation rate in about half of the CD4+ T-cell pool unambiguously shows that absolute CD4+ T-cell turnover is increased in the asymptomatic phase of immunodeficiency virus infection. At first glance, this would support the high turnover model. According to this model, initially brought up to explain the sharp rise in CD4 counts after initiation of antiretroviral therapy,8,9 the infection leads to increased death rates. As a result, CD4+ T-cell proliferation is augmented in a homeostatic effort of the immune system to maintain total CD4+ T-cell counts. However, according to this scenario, the immune system overshot the mark of just replacing lost CD4+ T cells in the group of asymptomatic animals of our study. Rather than by homeostatic mechanisms, the increased size of the CD4+ T-cell pool could be explained by chronic immune activation38,39 stimulated by the ongoing viral replication. The increased death rates for both subsets would then possibly be the result of a homeostatic mechanism to resolve the increased numbers of T cells. Additional support for the idea that the increased activation and proliferation of T-helper cells in HIV infection is caused by antigenic stimulation is provided by very recent studies.14,15,18,40,41 Because immune activation induces trapping of T cells in lymphoid organs42 and differentially influences the distribution of CD4+ and CD8+ T cells, this model could also explain the altered distribution of lymphocytes found in the SIV-infected macaques.24 The mechanisms for this global activation of T-cell subsets as well as of NK cells and B cells (data not shown and Rosenzweig et al11) are less clear. Both direct stimulation of virus-specific cells or indirect activation of bystander cells could contribute to this phenomenon. If antigenic stimulation is indeed the driving force behind the expansion of CD4+ T cells, a high number of virus-specific T-helper cells should be present. However, a T-helper cell response, as measured by in vitro antigen-specific proliferation, is absent in most HIV patients.43 HIV-specific blood T cells may be terminally differentiated effector cells no longer able to proliferate.44 Indeed, HIV-specific T-helper cells with effector function have been detected in blood at all stages of the infection with appropriate techniques.45 Possibly, proliferation of virus-specific cells occurs in lymphoid organs, which account for most of the increase in total T-cell numbers. Alternatively, some low level of antigen-specific proliferation could be amplified by unspecific stimulation of bystander cells as described for other viral infections.46 Interestingly, rapidly progressing animals, which do not mount a virus-specific immune response, also do not show increased activation and proliferation of CD4+ T cells in blood.29 Therefore, direct effects of the viral replication alone on the increased T-cell turnover such as viral antigens that drive cytokine production and CD4+ T-cell proliferation seem less likely. According to our data, the total number of CD4+ and CD8+ T cells does not expand as fast (doubling time 290 days and 130 days, respectively) as the increase in Ki-67 staining would suggest. Most likely the death rates were increased as well in the animals either by activation-induced cell death or by homeostatic mechanisms. In any case, the differences between increased proliferation rates and actual increment rates are similar for CD4+ and CD8+ T cells. This indicates that the same regulatory mechanisms are acting on both subsets. However, direct or immune-mediated killing of infected cells may additionally contribute to death of CD4+ T cells. Such effects could be responsible for the different pattern of correlation between viral load and proliferation rate of CD4+ and CD8+ T cells (Figure 8). In summary, the increased death rates found in HIV-infected patients and SIV-infected animals10-12,18 are the result of increased proliferation, rather than the cause, as was initially suggested.8,9,17 Our results show a biphasic course of T-cell turnover in immunodeficiency virus infection. An expansion of the total T-cell pool during the asymptomatic phase is followed by a decrease of T-cell numbers. What mechanisms upset the balance, ultimately causing immunodeficiency and death? A possible clue is provided by the almost complete loss of thymocytes in all animals with AIDS despite relative normal absolute CD4 counts in other organs. Because these monkeys were killed with the first signs of immunodeficiency, we think that this reflects the picture at the transition between the asymptomatic and the symptomatic stage of the infection. Possibly, either the chronic activation of the immune system or the unrestricted viral replication interferes with the regenerative capacity of the thymus or bone marrow precursors.1
We thank S. Czub, T. Kerkau, C. Hahn, B. Hofmann, F.-J. Kaup, N. Stolte, and E. Kuhn for help in the preparation of organs and cells, and J. Westermann, N. Stilianakis, and S. Coley for critical reading of the manuscript. We are indebted to F. Kirchhoff and U. Sauermann for providing us with material from animals used in their experiments.
Submitted June 5, 2002; accepted September 24, 2002.
Prepublished online as Blood First Edition Paper, October 10, 2002; DOI 10.1182/blood-2002-06-1644.
Supported by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Germany (BMBF 01 KI 9762/5, 01 KI 0211).
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.
Reprints: S. Sopper, Institut für Virologie und Immunbiologie, Julius- Maximilians-Universität, Versbacherstr 7, D-97078 Würzburg, Germany; e-mail: sopper{at}vim.uni-wuerzburg.de.
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Abstract
Introduction
