Paul-Ehrlich-Institute, Paul-Ehrlich-Str. 51-59, 63225 Langen, Germany1
Author for correspondence: Edgar Holznagel. Fax +49 6103 77 1234. e-mail holed{at}pei.de
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Abstract |
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Introduction |
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We have performed three-colour flow cytometric analyses to study the role of CD8+ T cells in SIVagm-infected AGMs and applied marker combinations (CD28, CD27, CD45RA and CD11a) shown to enumerate the whole effector CD8+ T cell subset. We also evaluated DP T cells in infected animals and determined their numbers in different subspecies. We included a population of animals living on one of the Caribbean islands (Barbados), which are known to be free from SIV (Daniel et al., 1988 ; Hendry et al., 1986
); the monkey population of Barbados phenotypically resemble African sabaeus monkeys. As these animals are the descendants of only a few, probably SIV-free (Daniel et al., 1988
; Lekutis & Letvin, 1995
), AGMs brought from the African continent to the Caribbean islands (Denham, 1981
) in the 17th century, it is likely that their immune systems have developed for several hundred years in the absence of SIV (Daniel et al., 1988
).
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Methods |
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Monoclonal antibodies (mAbs) and sample preparation.
The mAbs used for this study are listed in Table 1. Since no antibodies against CD19 with sufficient cross-reactivity have been found, B cell definition was restricted to the use of a mAb against CD20. For the purpose of our study, lymphocyte subsets were defined as follows: CD8 T cells (CD8
+CD3+CD4neg), DP T cells (CD4+CD3+CD8
+), single-positive (SP) CD4 T cells (CD4+CD3+CD8
neg) and natural killer (NK) cells (CD3negCD8
+, either CD16+ or CD16neg). Antibodies were diluted according to the manufacturers guidelines. Aliquots of 50 µl EDTA-anticoagulated blood from each animal were incubated with each of these reagent combinations for 20 min at room temperature before lysis and fixation using the Coulter Immunoprep Reagent system (Coulter), as described elsewhere (Macey et al., 1997
). Lysed and fixed samples were washed with 1·0 ml PBS solution (PBSS) and centrifuged for 5 min at 300 g. The supernatants were removed, cells were resuspended in 300 µl PBSS and analysed within 2 h.
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Determination of the virus load.
Peripheral blood lymphocytes (PBLs) were separated by FicollHypaque density centrifugation. After washing, PBLs were stored at -80 °C as 5x105 cell pellets for subsequent PCR studies. Cells were removed from the plasma by centrifugation and 500 µl aliquots frozen at -80 °C until used for quantitative RTPCR assays. RNA was isolated from 200 µl plasma using the High Pure Viral RNA kit (Roche), according to the manufacturers instructions, eluting in 50 µl RNAse-free water. DNA was isolated from 5x105 cells using the QIAamp DNA Blood Mini kit (Qiagen). The proviral and viral RNA loads were quantified as described previously (Holzammer et al., 2001 ).
Serology.
Titres of virus-specific antibodies were determined using standard ELISA protocols. Sera were diluted 1:100 in PBSS, 2% (w/v) milk powder and 0·05% (v/v) Tween 20 and were tested on both uncoated ELISA plates and plates coated with detergent-disrupted sucrose gradient-purified whole SIVagm. Sera were scored as positive when the OD value against antigen minus the OD value against empty plates was greater than 0·2.
Statistical analyses and data presentation.
The flow cytometric parameters of the groups were analysed for significant differences (P<0·05) by the MannWhitney test (PMW). Correlations were calculated and expressed as the Spearman coefficient of correlation (rs). Data were displayed using the box-and-whisker plot method. In all figures displaying box plots, the box extends from the 25th percentile to the 75th percentile, with a horizontal line at the median, and the whiskers extend down to the smallest value and up to the largest value.
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Results |
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The RNA virus load in the animals tested (n=18, mean age 6·2 years, range 2·59·1) ranged from 4·7x104 to 1·1x107 RNA copies/ml plasma (mean value 2·24x106). All animals were SIVagm-seropositive for at least 2 years.
When the haematological parameters (expressed as median, 2575th percentile and 1090th percentile) of infected (n=15 female animals, 5·5±1·6 years old) and uninfected (n=16) age- and sex-matched vervet monkeys were analysed by the MannWhitney test, increased absolute lymphocyte counts, expressed as x103 cells/µl blood, of 4·2, 3·25·5 and 3·07·5 (SIVagm+) versus 3·0, 2·34·4 and 1·75·5 (SIVagmneg) (PMW=0·05) and decreased platelet counts, expressed as x105 cells/µl blood, of 2·6, 2·33·3 and 2·13·7 (SIVagm+) versus 3·6, 2·95·2 and 2·56·9 (SIVagmneg) (PMW=0·01) were found in infected animals.
We determined the normal composition of PBL subsets by three-colour flow cytometric analysis of EDTA-anticoagulated whole blood samples from uninfected AGMs (Table 2). High numbers of DP T cells were found in AGM subspecies from the African continent but not in Caribbean sabaeus monkeys (Fig. 1
and Table 2
). DP T cells did not express the CD8
-chain, only the
-chain (Fig. 1
). Another striking difference between African and Caribbean AGMs concerned the population of double-negative (DN) T cells. This population accounted for, at most, 4% of T cells (Table 2
) in either infected or uninfected animals of African origin, but up to 22% of T cells in Caribbean sabaeus monkeys (Fig. 1
and Table 2
). Unfortunately, cross-reactive antibodies against common epitopes of simian
or
T cell receptor (TCR)-positive cells, which would allow the identification of DN T cells as TCR
+ or TCR
+, were not available. However, two mAbs recognizing subsets of human TCR
+ cells were found to cross-react with simian homologues. TCRV
1+ and TCRV
2+ PBLs were negative for CD4 and normally expressed the CD8
-chain in all AGM subspecies examined (data not shown).
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Finally, we examined the effect of long-term SIVagm infection on the composition of blood lymphocyte subsets (Figs 3
6
). Female SIVagm-infected vervet monkeys (n=17, 5·7±1·6 years old) and age- and sex-matched uninfected (n=17) vervet monkeys were selected randomly to determine the effect of SIVagm infection on the composition of blood lymphocyte subsets. A number of relative (Figs 3
and 5
) and absolute lymphocyte subset counts (Figs 4
and 6
) was significantly different between the two groups. Significantly higher absolute CD8
+ T cell numbers were found in the infected monkeys (2775±1510 cells/µl blood versus 1771±1160 cells/µl blood, PMW=0·03) (Fig. 4
). This was due mainly to a significantly higher level of CD8
+CD3+CD28neg PBLs [1094±986 cells/µl blood (Fig. 4
) versus 402±364 cells/µl blood (Fig. 5
), PMW=0·03]. When gated on CD8
bright+ PBLs, which were identical with CD8
+ T cells (Fig. 2d
), the absolute numbers of CD28negCD27neg cells within this CD8 subset were higher in SIVagm-infected AGMs (133±168 cells/µl blood) than in uninfected AGMs (39±90 cells/µl blood, PMW=0·0006) (Fig. 6
). Similar differences were found for the CD8
bright+CD28negCD11abright+ cell subset (197±255 cells/µl blood versus 83±170 cells/µl blood, P=0·02) (Fig. 6
), but not for the CD8
bright+CD28neg CD45RAbright+ cell counts (Fig. 6
). The absolute CD20+ cell counts were significantly higher in the infected animals (644±330 cells/µl blood versus 437±345 cells/µl blood, PMW=0·03) (Fig. 4
), as were the absolute counts of CD8
+CD3neg PBLs (692±561 cells/µl blood versus 323±197 cells/µl blood, PMW=0·05) (Fig. 4
). To confirm our results obtained from the female vervet monkeys, we investigated a larger number of samples collected from SIVagm-infected (n=55, 6·2±1·9 years old) and uninfected grivet, vervet and tantalus monkeys (n=48, 5·5±2·4 years old). However, it was not possible to match exactly the control group with regard to age, sex and subspecies. We could confirm significantly higher absolute CD8
+CD3+CD28neg counts in the infected group compared with the uninfected group (818±751 cells/µl blood versus 371±330 cells/µl blood, PMW=0·0005). Changes among other subsets were not significantly different (data not shown).
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Discussion |
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The most prominent differences between the AGM and the human immune systems are the physiologically high frequency of CD8 T cells and the finding that the majority of CD4+CD3+ PBLs coexpress the CD8 molecule (Kuroda et al., 1998
; Beer et al., 1998
; Murayama et al., 1997
, 1998). Surprisingly, Caribbean sabaeus monkeys had only a few DP T cells but large numbers of DN T cells. Our data suggested that the DN T cells may not belong to the TCR
+ cells, since subsets of
+ cells expressed the CD8
-chain in AGMs, similar to rhesus monkey TCR
+ cells (Gan et al., 1995
).
A further novel finding is the higher frequency of blood CD8 T cells expressing the -homodimer, in contrast to human CD8+ T cells, the majority of which express the
-heterodimer (Schmitz et al., 1999
).
We found significant changes with age in the proportion of different lymphocyte subsets, changes that were similar to those reported for ageing humans (Fagnoni et al., 1996 ; Hannet et al., 1992
; Sopper et al., 1997
).
The mean plasma RNA virus load in long-term SIVagm-infected animals was not significantly different from values published for asymptomatic HIV-1-infected individuals (Ogg et al., 1999 ) and naturally SIVsmm-infected sooty mangabeys (Rey-Cuille et al., 1998
). A more detailed description of the provirus and virus loads was published recently (Holzammer et al., 2001
).
The observation of significantly low platelet counts in long-term SIVagm-infected AGMs, which, until now, has not been reported, is corroborated by the fact that (i) SIVagm can also cause severe thrombocytopenia within 12 years after experimental infection in a heterologous host system (pig-tailed macaques) (Hirsch et al., 1995 ) and (ii) low platelet counts are found frequently in HIV-infected patients (Moses et al., 1998
).
HIV-1 infection of humans (Rosenberg et al., 1998 ) and SIVmac infection of rhesus macaques (Letvin et al., 1999
) induce a CD8+ T cell lymphocytosis caused mainly by CD28neg cells (Borthwick et al., 1994
; Monteiro et al., 1996
; Mugnaini et al., 1999
). Surprisingly, we found the same in long-term SIVagm-infected AGMs. However, there was no difference between uninfected and infected monkeys when the proportion of CD8 T cells expressing the
-homodimer and those expressing the
-heterodimer was compared (data not shown). In contrast, HIV-1-infection is associated with a significant drop in CD3+CD8
+ T cell counts (Schmitz et al., 1999
).
The CD8+CD28neg T cell subset was characterized further by the use of mAbs directed against CD27, CD11a and CD45RA. CD11a is the
L-subunit of the leukocyte function-associated molecule-1 (LFA-1) (Hamann et al., 1999
). LFA-1 is increased on memory T cells and mediates adhesion of CTLs to target cells and lymphocyte adhesion to endothelial cells, i.e. involved in lymphocyte trafficking (Hamann et al., 1999
). The function of CD27 is not clear, but prolonged stimulation of T cells switches off CD27 expression irreversibly (Hamann et al., 1999
). CTLs were shown to be CD27neg but to re-express CD45RA and to be stained brightly with CD11a (Hamann et al., 1999
). HIV-1 (Dalod et al., 1999
; Giorgi et al., 1999
; Hamann et al., 1999
; Ogg et al., 1999
; Lewis et al., 1999
; Mugnaini et al., 1999
; Posnett et al., 1999
; Weekes et al., 1999
) and SIVmac-specific CTLs (Kuroda et al., 1998
) are found among these CD3+CD8
+CD28negCD27negCD11abright+ cells. It is very likely that this subset also comprised the CTL subset in AGMs. However, it is not yet possible to functionally characterize and enumerate virus-specific CTLs in AGMs.
Significantly increased NK and B cell counts have not been described previously in SIVagm-infected AGMs. Interestingly, progression towards AIDS is associated with decreasing NK cell numbers, both in HIV-1 (De Souza et al., 2000 ; Margolick et al., 1991
; Peruzzi et al., 2000
; Vuillier et al., 1988
) and in SIVmac infection (Powell et al., 1989
; Vowels et al., 1990
).
Taking the virological, haematological and immunological data together, there are at least three common characteristics of the natural SIVagm system and the heterologous lentivirus systems: comparable plasma virus loads, thrombocytopenia and an increase in CD8+CD28neg T cell counts. In contrast to pathogenic lentivirus infections, however, we found significantly elevated NK cell numbers and normal absolute CD4 T helper cell counts in infected AGMs. Despite the lack of overt disease progression, the lowered platelet counts suggest that AGMs are not completely protected from the deleterious effects of SIVagm infection.
Lentiviruses can prevent CTL-mediated killing of infected cells by a nef-mediated downregulation of MHC-I molecules (Piguet et al., 1999 ) and concurrently lowered NK cell numbers allow the escape from nonMHC-restricted cytotoxicity (Powell et al., 1989
; Vowels et al., 1990
). Although it has yet to be demonstrated formally that the SIVagm Nef protein is able, like that of HIV, to downregulate expression of MHC-I molecules, elevated NK cell numbers may contribute to the natural resistance in AGMs.
In pathogenic lentivirus infections, CTLs appear to be effective in the short- and medium-term containment of the virus but are ultimately unable to control the infection (Goulder & Walker, 1999 ). As innate immunity has the function of nonMHC-restricted killing of infected cells (Fearon & Locksley, 1996
; Medzhitov & Janeway, 1997
; Peruzzi et al., 2000
) and is involved in the initiation of adaptive immune responses (Fearon & Locksley, 1996
; Levy, 2001
; Medzhitov & Janeway, 1997
) and secretion of chemokines (Levy, 2001
), it is tempting to hypothesize that virus escape mutants (Evans et al., 1999
) may be recognized more effectively by the adaptive arm of the immune system in long-term infected AGMs, whereas the decrease in NK cell numbers during progression towards human and simian AIDS may support the generation of newly produced SIV variants, at least due to a lower interferon-
concentration produced by NK cells (Peruzzi et al., 2000
) and lower chemokines (Levy, 2001
). Similarly, the loss of CD4 T cells in AIDS patients also impairs the generation and function of newly produced CTLs (Goulder & Walker, 1999
). Since the absolute CD4 T cell counts are stable in long-term-infected AGMs, one may argue that this may allow the maintenance of an effective CTL response. A third possible explanation for the persistent CTL response in AGMs is the maintenance of lymph node architecture throughout the course of infection (Beer et al., 1996
), which would enable the immune system to continue producing CTLs by peripheral expansion. Despite their elevated levels, there was no evidence that the CD8 T and NK cells contributed to a lower RNA virus load and we cannot exclude the possibility that the high CTL counts observed resulted from unspecific bystander activation (Tough & Sprent, 1996
; Welsh et al., 2000
).
To conclude, long-term SIVagm infection of its natural AGM host, although inducing no signs of overt AIDS, does have profound effects on the immune system of the animal. Significant increases in cells of the NK, CTL and B cell phenotypes indicate continuous and vigorous immune activation. Whether or not these cells, or indeed the naturally high level of CD4/CD8 DP cells in circulation, play a decisive role in preventing disease remains to be elucidated.
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Acknowledgments |
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Footnotes |
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Received 4 September 2001;
accepted 2 November 2001.