HIV-1 transgenic rats develop T cell abnormalities
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Transcript of HIV-1 transgenic rats develop T cell abnormalities
www.elsevier.com/locate/yviro
Virology 321 (2004) 111–119
HIV-1 transgenic rats develop T cell abnormalities
William Reid,a,* Sayed Abdelwahab,b,c Mariola Sadowska,a David Huso,d Ashley Neal,f
Aaron Ahearn,b,e Joseph Bryant,f Robert C. Gallo,a George K. Lewis,b,c and Marvin Reitza,c
aDivision of Basic Science, University of Maryland, Baltimore, MD 21201, USAbDivision of Vaccine Research, University of Maryland, Baltimore, MD 21201, USA
cDepartment of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201, USAdDivision of Comparative Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
eMolecular and Cellular Biology Program, University of Maryland, Baltimore, MD 21201, USAfAnimal Model Division, Institute of Human Virology, University of Maryland, Baltimore, MD 21201, USA
Received 3 September 2003; returned to author for revision 2 December 2003; accepted 4 December 2003
Abstract
HIV-1 infection leads to impaired antigen-specific T cell proliferation, increased susceptibility of T cells to apoptosis, progressive
impairment of T-helper 1 (Th1) responses, and altered maturation of HIV-1-specific memory cells. We have identified similar impairments in
HIV-1 transgenic (Tg) rats. Tg rats developed an absolute reduction in CD4+ and CD8+ T cells able to produce IFN-g following activation
and an increased susceptibility of T cells to activation-induced apoptosis. CD4+ and CD8+ effector/memory (CD45RC�CD62L�) pools were
significantly smaller in Tg rats compared to non-Tg controls, although the converse was true for the naı̈ve (CD45RC+CD62L+) T cell pool.
Our interpretation is that the HIV transgene causes defects in the development of T cell effector function and generation of specific effector/
memory T cell subsets, and that activation-induced apoptosis may be an essential factor in this process.
D 2004 Elsevier Inc. All rights reserved.
Keywords: Th1/Th2 cells; HIV-1; Transgenic
Introduction
In nonprogressive HIV-1 infection, both T-helper (Th)
and cytotoxic T-lymphocyte (CTL) responses appear to be
maintained. However, as HIV-1 disease progresses, there is
a decline in the number of CD4+ T cells and a loss of HIV-
1-specific Th and CTL responses (Kalams et al., 1999;
Rosenberg et al., 1997). With advanced HIV-1 disease,
there are not only losses in specific helper and CTL
responses to HIV-1 antigens, but also a loss of immune
responses to opportunistic pathogens such as cytomegalo-
virus (CMV) (Komanduri et al., 2001). The loss of helper
activity is likely to be an important factor in the decline of
CTL activity. Furthermore, the ability to eradicate HIV-1
infection by CTL-mediated lysis is clearly undermined
0042-6822/$ - see front matter D 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.virol.2003.12.010
Abbreviations: Th, helper CD4 T Cell; Tc, helper CD8 T Cells; HIV,
human immunodeficiency virus; Tg, transgenic.
* Corresponding author. Division of Basic Sciences, Institute of Human
Virology, University of Maryland Baltimore, 725 West Lombard Street,
Baltimore, MD 21201. Fax: +1-410-706-4694.
E-mail address: [email protected] (W. Reid).
during the chronic course of the disease by impairment
of both Th1 and type 1 CD8+ T cell (Tc1) effector
responses (Kaech and Ahmed, 2003; Shedlock and Shen,
2003; Sun and Bevan, 2003).
Memory T lymphocytes provide rapid and direct immu-
nological protection against diseases. Their formation
depends upon T cell expansion following initial encounter
with antigen, the acquisition of effector function (secretion
of cytokines), a contraction or ‘‘death phase’’ during which
effector cell numbers are reduced, and finally the develop-
ment of memory (Ahmed and Gray, 1996; Murali-Krishna et
al., 1998). Memory CD4+ and CD8+ T cells can quickly
expand and acquire effector function following reexposure
to antigens (Ahmed and Gray, 1996; Murali-Krishna et al.,
1998). Alteration of any phase of these T cell responses can
affect the number of memory T cells that are formed and
determine whether protective immunity is established
(Kaech et al., 2002).
CD4+ T-helper lymphocytes are classified as Th1 or
Th2 cells based on their patterns of cytokine production.
Th1 cells produce IFN-g, IL-2, and tumor necrosis factor
(TNF)-h, promote the expansion of CTLs, and promote
Fig. 1. Tg rat PBMCs produce reduced levels of IFN-g. Production of
extracellular IFN-g from PMA-I activated PBMCs from 12- to 15-month-
old Tg (n = 3), and non-Tg rats (n = 3) were assayed by capture ELISA as
described in Materials and methods. The times after stimulation of the cell
at which assays were performed are indicated. Error bars indicate standard
deviations. *Indicates a significant difference.
W. Reid et al. / Virology 321 (2004) 111–119112
secretion of IgG2a, opsonizing and complement-fixing
antibodies by B cells (Seder and Paul, 1994). In addition,
they participate in macrophage activation and delayed-type
hypersensitivity (DTH) reactions (Seder and Paul, 1994).
Th2 cells produce IL-4, IL-5, IL-6, and IL-10, provide
help primarily for humoral immune responses, promote
secretion of IgG1 and IgE antibodies by B cells, and
inhibit CTLs and several macrophage functions (Seder
and Paul, 1994). CD8+ T lymphocytes are also classified
as distinct effector cell types based on their patterns of
cytokine expression. Tc1 expresses IFN-g and kills target
cells by either perforin or Fas-mediated mechanisms. Type
2 CD8+ cells (Tc2) express IL-4 and kill target cells by the
perforin pathway only (Carter and Dutton, 1995; Sad et al.,
1996).
We have previously described a transgenic (Tg) rat that
contains a gag-pol-deleted HIV-1 provirus (Reid et al.,
2001). The HIV-1 Tg rat expresses HIV viral RNA and
proteins in lymphocytes and monocytes and develops
apparent defects in Th1 immune responses, as evidenced
by a reduced delayed-type hypersensitivity (DTH) to
keyhole limpet hemocyanin (KLH). The lymph tissue from
these Tg rats manifests an AIDS-like histopathology,
including depletion of lymphocytes within the T cell
region of the spleen and a B cell follicular hyperplasia.
Here we present a more detailed analysis of immune
functions in the HIV-1 Tg rat that shows that there are
defects in both CD4+ and CD8+ T cell effector function
and effector/memory development and an increase in
activation-induced apoptosis of T cells. Thus, the Tg rat
is potentially a useful small animal model to investigate
how HIV infection affects the acquisition of effector/
memory T cells.
Results
HIV-1 Tg rat T lymphocytes produce reduced levels of
IFN-c
The defective DTH reaction to KLH recall antigen by
HIV-1 Tg rats (Reid et al., 2001) led us to ask whether this
was correlated with defective production of type-1 cyto-
kines. Therefore, we compared production of IFN-g in
vitro by phorbol 12-myristrate 13-acetate and ionomycin
(PMA-I) treated peripheral blood mononuclear cells
(PBMCs) from mature (12–15 months old) Tg and control
rats (n = 3) by capture ELISA of culture supernatants as
described in Materials and methods. Fig. 1 shows that
IFN-g production from Tg rat PBMC was indeed signif-
icantly decreased (127 F 95 pg/ml at 5 h and 536 F 388
pg/ml at 18 h) after stimulation relative to that of PBMC
from age-matched controls (530 F198 and 2094 F 622
pg/ml). The approximate fourfold reduction in IFN-g
production by HIV-1 Tg rat PBMC was significant at both
time points (P < 0.05 and P < 0.04, respectively),
suggesting that production of IFN-g is inhibited or that
the development of specific effector/memory cell popula-
tions is impaired.
HIV-1 Tg rats have reduced numbers of CD4+ and CD8+
peripheral blood T lymphocytes expressing IFN-c following
in vitro activation
We wanted to determine whether the levels of IFN-g in
culture supernatants were lower because fewer T cells from
Tg rats produced IFN-g or because less IFN-g is produced
by the same number of IFN-g positive T cells. PMA-I
stimulated PBMCs from mature (12–15 months) Tg, and
control rats were analyzed for intracellular IFN-g by flow
cytometry as described in Materials and methods. Results
from individual representative Tg rats (Figs. 2A, B) show
that 1.4% and 9.0% of Tg CD4+ cells and CD8+ cells
expressed IFN�g, compared to values for an age-matched
control of 7.9% and 22.2%. The mean channel fluores-
cence intensity for intracellular IFN-g production was 5.4
and 10.6 for Tg CD4+ and CD8+ T cells, compared with
20.6 and 24.5, respectively, for age-matched controls Figs.
2A, B. Fig. 2C averages the results from multiple experi-
ments. A mean of 2.1 F 1.6% CD4+ (n = 5) and 7.3 F1.8% CD8+ (n = 4) T cells from Tg rats expressed
intracellular IFN-g, compared with 9.5 F 2.5% CD4+
(n = 5; P < 0.03) and 17.7 F 6.9% CD8+ (n = 4; P <
0.03) T cells from controls. A significant reduction (P <
0.03) in intracellular IFN-g production from CD4+ T cells
and a twofold reduction in CD8+ T cells were also seen in
young (3–6 months old) Tg rats compared to age-matched
controls (data not shown). This suggests that reduced
production of IFN-g by Tg PBMC is due at least in part
to reduced numbers of cells able to produce IFN-g and that
Fig. 2. Tg rats have reduced numbers of T cells producing IFN-g. Intracellular IFN-g production in peripheral blood T cells from Tg and non-Tg control rats is
shown. PBMCs from 12- to 15-month-old Tg and non-Tg HIV-1 control rats were analyzed for intracellular IFN-g by flow cytometry following PMA-I
stimulation as described in Materials and methods. A representative experiment is shown in the two top panels (A, B). Panel C shows the average intracellular
staining for IFN-g with CD4+ (n = 5) and CD8+ (n = 4) lymphocytes from multiple Tg (open bars) and normal control (filled bars) rats as indicated. Differences
between both CD4+ and CD8+ Tg T cells versus age-matched controls were statistically significant ( P < 0.03). The numbers represent the mean values F the
standard deviation. *Indicates a significant difference.
W. Reid et al. / Virology 321 (2004) 111–119 113
young and mature Tg rats have a similar defect in IFN-g
production. There was also a significant reduction in mean
channel fluorescence intensity for intracellular IFN-g pro-
duction in T cells of both young (not shown) and mature
Tg rats, suggesting that reduced levels of expression by
IFN-g positive T cells also contributes to decreased IFN-g
production.
In contrast with IFN-g, intracellular expression of a
representative Th2 cytokine (IL-10) did not significantly
differ between Tg and non-Tg controls following short-term
stimulation with PMA-I; additionally, end-point titers for
IgG1 did not significantly differ between Tg and non-Tg
controls 4 weeks following intraperitoneal immunization
with 100 Ag of KLH in complete Freunds’s (not shown).
These data suggest that the dysregulation in Th2 effector
function is not as severe as the Th1 dysregulation. Mature
Tg rat T cells from a second HIV-1 Tg line, which contains a
lower transgene copy number in a different insertion site
(Reid et al., 2001), also had significantly (P < 0.05) fewer T
cells positive for intracellular IFN-g than did non-Tg con-
trols [4.1 F 0.7% (n = 3) of CD4+ and 2.2% F 0.21% (n =
3) of CD8+ compared with 9.9 F 1.9% (n = 7) and 15.06 F6.8% (n = 6) for controls}. No significant differences in
intracellular IFN-g expression between animals Tg for an
irrelevant gene and controls were observed (data not
shown). These data suggest that the observed phenotype is
not a positional effect mediated by mutagenesis of a cellular
gene.
Defective development of effector/memory CD4+ and CD8+
T cell subsets in HIV-1 Tg rats
The reduced numbers of Tg rat T cells that express IFN-g
suggest that generation or maintenance of effector/memory
cell populations is defective. We therefore asked whether the
reduced proportion of T cells able to express IFN-g is
correlated with a reduction in the size of the memory/
effector T cell pools. CD4+ T cells in the rat are classified
into four phenotypic subpopulations based on the expres-
sion of CD45RC and CD62L. The CD45RC+CD62L+
phenotype defines naı̈ve cells (Hylkema et al., 2000;
Ramirez and Mason, 2000). The activation of naı̈ve
CD45RC+CD62L+ lymphocytes leads to downregulation
of CD45RC and CD62L and progressive distribution to
distinct CD45RC�CD62L+, CD45RC�CD62L�, and
CD45RC+CD62L� effector/memory subsets (Powrie and
Mason, 1990; Ramirez and Mason, 2000).
To examine these four phenotypic subsets, freshly iso-
lated, nonstimulated CD4+ and CD8+ peripheral blood T
cells from young and mature Tg and non-Tg controls were
analyzed by flow cytometry as described in Materials and
methods. These naturally occurring memory cells are
thought to be generated against environmental antigens
found in gut flora and food. Naı̈ve (CD45RC+CD62L+)
and effector/memory (negative for CD45RC, CD62L, or
both) subset distributions were determined and compared
between Tg and non-Tg control rats. Fig. 3 shows the
Fig. 3. Development of effector/memory CD4+ and CD8+ T cell subsets is impaired in HIV-1 Tg rats. Representative flow cytometric analyses are shown for CD4+ and CD8+ profiles gated on CD3 T cells (A, B),
naı̈ve (CD45RC+/CD62L+) and effector/memory (CD45RC+/CD62L�, CD45RC�/CD62L�, and CD45RC�/CD62L+) subsets in mature Tg and control rats (C–F). Panels a and b show the CD4 and CD8 profiles
for Tg and non-Tg, respectively. Panels C and E show the subset distributions for CD4+ and CD8+ T cells, respectively, for a non-Tg control. The subset distributions for CD4+ and CD8+ T cells from a Tg rat are
shown in panels D and F, respectively. The bold numbers in each quadrant indicate the percent of the total cell population present in that quadrant. Panels G and H summarize results from multiple experiments
measuring the distribution of naı̈ve and effector/memory cell phenotypic subsets of CD4+ and CD8+ T cells, respectively, from young (3–6 months old) Tg (n = 6) and control rats (n = 5). Panels I and J show data
from similar experiments with CD4+ and CD8+ T cells, respectively, from mature (12–15 months old) Tg rats (n = 6) and age-matched non-Tg controls (n = 8). The numbers represent the mean values F the
standard deviation. *Indicates a significant difference.
W.Reid
etal./Viro
logy321(2004)111
–119
114
W. Reid et al. / Virology 321 (2004) 111–119 115
percentages of total CD4+ and CD8+ cells within each
population. Panels (a) and (b) show the CD4 and CD8
profiles for Tg and non-Tg, respectively. Representative
distributions of naı̈ve and effector/memory cell subsets in
a mature Tg rat and a non-Tg control rat are shown in Figs.
3C–F. As shown in Figs. 3G, H, the mean percentages of
CD4+ and CD8+ with naı̈ve and effector/memory cell
surface phenotypes did not significantly differ between
young (3–6 months old) Tg and non-Tg control rats.
However, in older animals (12–15 months old) (Fig. 3I),
the most abundant subpopulation of CD4+ cells (61%) from
control rats was that of the CD45RC�CD62L� effector/
memory phenotype, and the size of this population was
significantly smaller in age-matched Tg rats (P = 0.007). In
contrast, the most abundant CD4+ subpopulation in Tg rats
(55%) was that with a CD45RC+CD62L+ naı̈ve phenotype,
which was significantly larger that that from age-matched
controls (P = 0.007). Similarly, the CD8+ subpopulation of
the CD45RC�/CD62L� effector/memory phenotype was
significantly decreased ( P = 0.007) and that of the
CD45RC+/CD62L+ naı̈ve phenotype significantly increased
(P = 0.003) in older Tg rats (Fig. 3J) relative to age-matched
controls (4% and 79%, respectively).
These data show that the distribution of CD4+ and CD8+
T cells from mature Tg rats between the CD45RC+CD62L+
naı̈ve phenotype and CD45RC�CD62L� effector/memory
phenotype pools is perturbed compared to age-matched non-
Tg controls. It has been previously reported that effector/
memory T cell populations are maintained by homeostatic
proliferation, which depends upon the cytokine microenvi-
ronment (Kaech et al., 2002; Murali-Krishna et al., 1998;
Seddon and Zamoyska, 2003; Seddon et al., 2003). Al-
though the complements of cytokines that regulate the
generation and survival of effector/memory CD4+ and
CD8+ T cells are not completely characterized, our data
indicate that the CD45RC�CD62L� subpopulation is not
generated and maintained in the mature HIV-1 transgenic
animals.
Total CD4+ and CD8+ peripheral blood lymphocyte
absolute numbers in Tg and control rats are similar
It was of obvious interest to determine whether differ-
ences in absolute T cell numbers could help explain the
Table 1
Absolute lymphocyte numbers and percentages of lymphoid subsets in periphera
Percent CD4+
T cellsaPercent CD8+
T cellsa
Transgenic, n = 4 26 F 7 11 F 2
Control, n = 4 37 F 10 13 F 3
a Percentages of CD4+ and CD8+ T cells from PBMCs were determined by flow c
per microliter of blood.b Mean absolute numbers of peripheral blood lymphocytes from Tg and non-Tg
*There was a statistical difference ( P < 0.05) in the mean lymphocyte number pe
Student’s t test.
differences between Tg rats and controls in the numbers of T
cells able to express IFN-g or in the relative sizes of effector/
memory and naive T cells subsets. We therefore determined
the percentages of CD4+ and CD8+ lymphocytes from
PBMCs of mature Tg and control rats by flow cytometry
and calculated the mean absolute numbers of CD4+ and
CD8+ T cells from the mean number of lymphocytes per
microliter of blood as described in Materials and methods.
Interestingly, the mean number of lymphocytes per microli-
ter of blood was significantly elevated in Tg rats (P < 0.05;
Table 1). However, there were no significant differences in
the mean absolute numbers of CD4+ and CD8+ T cells per
microliter of blood. In a separate set of experiments, B
lymphocyte percentages (detected as CD3�CD45RA/B+)
in mature Tg rats were significantly (P < 0.05) increased
compared with age-matched controls (data not shown), and
this accounts for the differences in total lymphocyte numb-
ers. These data suggest that in mature transgenic rats, there is
a lack of effector/memory CD4+ and CD8+ T cell with the
CD45RC�CD62L� phenotype, which is followed by a
homeostatic expansion of the CD45RC+CD62L+ naı̈ve sub-
population. Alternatively, there could be a block in lineage
differentiation to effector/memory that causes an accumula-
tion of naı̈ve (CD45RC+CD62L+) T cells. This impairment
appears to increase with age, although the defect in IFN-g
expression is already apparent in CD4+ T cells even in
younger animals (Fig. 2).
Increased activation-induced apoptosis of CD3+
lymphocytes from Tg rats
One mechanism that could lead to a reduced generation
of effector/memory cells is an increased susceptibility to
apoptosis of activated T cells. We therefore asked whether T
cells from mature Tg rats were more susceptible to in vitro
activation-induced apoptosis than those from non-Tg con-
trols. PBMC were stimulated with PMA-I as described in
Materials and methods, and apoptosis was measured by
staining CD3+ positive lymphocytes for surface exposure of
Annexin V. Figs. 4A, B show representative Annexin V
staining in stimulated and nonstimulated CD3+ T cell
populations. Fig. 4A shows that 5 h following PMA-I
stimulation, Annexin V incorporation was 29.6% and
37.6% for non-Tg control and Tg rats, respectively. Fig.
l blood from mature Tg rats and age-matched controls
Mean number
lymphocytes/AlbMean number
CD4+/AlaMean number
CD8+/Ala
7636 F 1688* 1985 F 534 840 F 153
3806 F 690* 1408 F 381 494 F 114
ytometry and their absolute numbers calculated from the mean lymphocytes
controls were determined using a Cell-Dyn Coulter counter.
r microliter of blood between Tg and control animals, as determined by the
Fig. 4. Increased activation-induced apoptosis of Tg rat T cells. Mature (12–15 months old) transgenic (n = 5) and age-matched control (n = 4) PBMCs were
stimulated for 2.5 and 5 h with PMA-I. Apoptosis was determined at the indicated times as the difference from stimulated and nonstimulated in CD3+
lymphocytes in the percent of Annexin V incorporation. Panels A and B show representative Annexin V staining for stimulated and nonstimulated populations,
respectively. Panel C show the percent change in Annexin V incorporation. The numbers represent the mean values F the standard deviation. *Indicates a
significant difference.
W. Reid et al. / Virology 321 (2004) 111–119116
4B shows that during the same time, Annexin V incorpo-
ration for nonstimulated populations were 12.7% and 8.4%
for non-Tg control and Tg rats, respectively. Fig. 4C shows
that 2.5 h after stimulation, the mean percent change in
Annexin V incorporation was similar (17.4 F 6.4 and 15.6
F 1.8) for Tg and control animals, respectively. Five hours
after stimulation, however, the mean percent change in
Annexin V incorporation in Tg PBMC was significantly
greater than that of control PBMC (24.7 F 4.5 vs. 14.4 F3.7, P < 0.05). These data suggest that increased apoptosis
of CD3+ cells contributes to the skewed distribution of
memory/effector cells in HIV Tg rats.
Discussion
We previously reported that HIV-1 Tg rats have impaired
Th1 immunity, as evidenced by a reduced DTH reaction to
KLH recall antigen (Reid et al., 2001). In the current study,
we have further established and characterized Th1 defects in
these animals. Following a 5-h PMA-I activation, type 1
cytokine (IFN-g) production by PBMCs was significantly
reduced compared to normal controls. This was reflected by
both a reduction in the numbers of CD4+ and CD8+ T cells
expressing IFN-g and reduced expression levels by IFN-g
positive cells (Figs. 1 and 2) and was evident for both young
and mature Tg rats. These data suggest a dysregulation in
the development of Th1 effector function, as well as a more
specific defect in IFN-g production. Consistent with the idea
that there is a selective defect in the development of
effector/memory cells, HIV Tg rats developed a deficit in
the numbers of CD4+ and CD8+ cells with a effector/
memory surface phenotypes and a reciprocal increase in
the relative and absolute numbers of CD4+ and CD8+ cells
with a naı̈ve phenotype (Fig. 3). Sequestration of effector/
memory T cells in lymphoid organs is not a likely explana-
tion of our results because we have previously reported that
lymph nodes in the transgenic rats were characterized by
W. Reid et al. / Virology 321 (2004) 111–119 117
lymphoid depletion and the transgenic rats also have marked
lymphoid depletion in the periarteriolar lymphoid sheaths in
the spleen, a site where T lymphocytes would normally
localize (Reid et al., 2001).
The hallmarks of specific T cell immunity include an
antigen-specific proliferative expansion, an acquisition of
effector function, an apoptotic contraction phase, and the
generation of memory (Ahmed and Gray, 1996; Murali-
Krishna et al., 1998). T cells from HIV-1 infected individ-
uals have enhanced rates of in vitro activation-induced
apoptosis compared with normal T cells, possibly due to
increased anergy (Groux et al., 1992; Meyaard et al., 1992).
Apoptosis in vivo following activation may be responsible
in part for loss of CD4+ and CD8+ T cell effector functions
in HIV-infected people (Lieberman et al., 2002). We have
previously shown that apoptosis of splenocytes in vivo from
Tg rats was significantly elevated compared with age and
sex matched controls (Reid et al., 2001). Here we show that
peripheral T cells from Tg rats also have an increased
susceptibility to activation-induced apoptosis in vitro, which
may help explain both the low numbers of peripheral blood
CD4+ and CD8+ T cells able to produce IFN-g and the
reduced generation of CD4+ and CD8+ cells with the
CD45RC�CD62L� effector/memory phenotype that
becomes apparent over time.
The abnormally large populations of CD4+ and CD8+ T
cells expressing the naı̈ve CD45RC+CD62L+ phenotype
that accumulate in mature Tg rats (Fig. 3) are striking. It
has been reported that during the generation of memory,
constant T cell numbers are maintained by homeostatic cell
proliferation (Kaech et al., 2002; Murali-Krishna et al.,
1998), and that T cells receiving weak or inadequate antigen
stimulation are programmed to die because they express low
levels of anti-apoptotic molecules and are less responsive to
homeostatic cytokines (Gett et al., 2003). Taken along with
the paucity of Tg effector/memory T cells with a
CD45RC�CD62L� phenotype, it is tempting to speculate
that weak stimulation of the Tg rat T cell receptor causes a
selective depletion or an atypical differentiation of effector/
memory T cells followed by a compensatory expansion of
the naı̈ve pool. These data contrast with those reported for
humans and mice that show that naı̈ve CD4+ T cell numbers
decrease because of thymus involution or HIV-1 infection
(Douek et al., 2001). However, recent reports show that the
quantity and quality of CD8+ T cells depends on CD4+ help
(Bourgeois et al., 2002; Janssen et al., 2003; Shedlock and
Shen, 2003; Sun and Bevan, 2003); the ability of memory
CD8+ T cells to produce IFN-g and to survive when
restimulated in the absence of help is reduced compared
to when help is provided (Bourgeois et al., 2002; Janssen et
al., 2003; Shedlock and Shen, 2003). HIV-1 gene products
have been shown to induce anergy (Lieberman et al., 2002),
apoptosis (Cohen and Fauci, 2 A.D.; Groux et al., 1992;
Roos et al., 1995), B cell activation (Berberian et al., 1993),
dysregulation of immune homeostasis (Tanchot et al., 1997),
and inhibition of Th1 immunity (Mirani et al., 2002).
Expression of one or more HIV proteins in the Tg rat likely
leads to anergy, apoptosis, suppression of Th1 responses,
and compensatory homeostasis of naı̈ve CD4+ and CD8+ T
cells, all of which may contribute to the skewed maturation
of naı̈ve into effector/memory T cells.
The observed immune irregularities raise some issues.
First, which viral gene product(s) is involved in the ob-
served immune irregularities? The gag and pol genes are
functionally deleted in the transgene in these Tg rats (Reid
et al., 2001), but all the other viral genes are present. In the
HIV Tg mouse, expression of Nef has been reported to
cause immune dysfunction (Hanna et al., 2001; Simard et
al., 2002), but whether this is the case in the HIV Tg rat is
not clear. We are currently constructing HIV-1 Tg rats with
knockouts of other viral genes to address this question, but
as of yet we have no data on specific genes. Second,
lymphocyte abnormalities of Tg rats may not be limited to
T cells. The older Tg rats have elevated peripheral blood B
cell counts, and we previously showed that spleen tissue
sections from Tg rats show an expansion of the B cell areas
(Reid et al., 2001). It will be interesting to determine the
functional and phenotypic properties of these B-lympho-
cytes. Third, it will be important to determine whether the
reduced size of the effector/memory pool is the result of
suboptimal priming and T cell expansion, selective deple-
tion of cells from the effector/memory pool, or a dysregu-
lation in lineage development of effector/memory T cells.
In summary, we describe a range of T cell abnormalities
in HIV-1 Tg rats that include an apparent dysfunction in the
generation of Th1 and Tc1 cells, as judged by IFN-g
production, an abnormal distribution of CD4+ and CD8+ T
cell subsets with naı̈ve and effector/memory phenotypes,
and an increased susceptibility of activated T cells to
apoptosis. Although these abnormalities are not entirely
identical to the immune irregularities seen in human HIV-
1 infection, HIV Tg rats represent a potentially useful small
animal model to investigate some of the immunopathologic
effects of HIV-1 gene products and their effects on the
generation of specific effector/memory T cells and immune
function.
Materials and methods
HIV-1 Tg and non-Tg animals
The construction of the transgene and production of the
transgenic rats have been described (Reid et al., 2001).
Young (3–6 months old) and mature (12–15 months old)
specific pathogen-free (SPF) Tg rats and age-matched
Fisher 344/NHsd non-Tg rats were used in our analyses;
these animals were housed under pathogen-free conditions
in microisolator cages on HEPA filtered ventilated racks.
The University of Maryland Institute of Biotechnology
Animal Care and Use Committee approved the experimental
protocol.
logy 3
Isolation of peripheral blood mononuclear cells
PBMCs were prepared from 1.0 ml of blood from 3- to
6- and 12- to 15-month-old rats in EDTA tubes. The blood
was diluted 1:1 with PBS, layered over 3.0 ml of Histo-
paque 1083 (Sigma, St. Louis, MO), and centrifuged at
400 � g for 30 min at room temperature. PBMCs were
collected, washed twice with PBS, and cultured as de-
scribed below.
Analysis of IFN-c in culture supernatants
PBMCs (1.0 � 106/ml) from Tg and control rats
were stimulated as described below, and their super-
natants were collected at 5, 18, and 24 h. Media were
collected and analyzed in triplicate. IFN-g levels were
measured using an ELISA cytokine detection kit (R&D
Systems, Minneapolis, MN) according to the manufac-
turer’s protocol.
Analysis of intracellular cytokines
Expression of surface and intracellular proteins was
assessed by four-color flow cytometric analysis. For
analysis of cytokine production by intracellular staining,
1.0 � 106 cells/ml PBMCs from Tg and normal control
animals were examined. Briefly, PBMCs cultured in
RPMI media 1640 supplemented with 10% heat-inacti-
vated fetal bovine serum (GIBCO, Grand Island, NY)
were stimulated for 2 h with 25 ng/ml of phorbol 12-
myristrate 13-acetate and 1 Ag/ml of ionomycin (PMA-I),
treated with GolgiStop [Beckton Dickerson (BD) Bio-
sciences, San Jose, CA], and cultured in 5% CO2 at 37
jC for an additional 4 h. At the appropriate time, cells
were stained with the primary antibody or an appropriate
isotype control according to the manufacture’s instructions
(PharMingen, San Diego, CA). For CD4+ and CD8+ T
cell subsets, cells were surface stained with APC anti-rat
CD4 (OX-35, PharMingen) and PerCP anti-rat CD8 (OX-
8, PharMingen). Following surface staining, cells were
fixed, permeabilized, and stained with PE anti-IL-10 (A5-
4, PharMingen) or FITC anti-IFN-g (DB-1, PharMingen)
according to the manufacturer’s recommendations. Sam-
ples were analyzed using a FACSCalibur (BD Bioscien-
ces) flow cytometer, and the data were analyzed by
CellQuest (BD Biosciences) or FlowJo software (Tree
Star, Inc., San Carlos, CA).
Analysis of naı̈ve and memory T cell subsets
Naı̈ve and memory T cell subsets, cells were ana-
lyzed by surface staining with PE anti-rat CD45RC
(OX-22, PharMingen), FITC anti-rat CD62L (HRL-1,
PharMingen), PerCP anti-rat CD8, and APC anti-rat
CD3 (1F4, PharMingen) as described by the manufac-
ture (PharMingen).
W. Reid et al. / Viro118
Complete blood count and determination of CD4+ and
CD8+ T cell subpopulations and B lymphocytes absolute
numbers
Blood from mature Tg and control rats was collected
into EDTA tubes and analyzed on a Cell-Dyn 3500R
Coulter counter (Abbott Laboratories, Abbott Park, IL)
for complete blood count (CBC) analysis. PBMCs were
collected as previously described and surface-stained
according to the manufacturer’s staining protocol with
PerCP anti-rat CD8, APC anti-rat CD3, or FITC anti-rat
CD45RA/B (MRC OX33, Cedarlane, Hornby, Ontario,
Canada). The CD8+ and CD4+ T cell populations were
characterized as CD3+ CD8+ and CD3+ CD8�, respective-
ly. B lymphocytes were characterized as CD3�CD45RA/
B+. The subpopulations were determined on a FACSCali-
bur (BD Biosciences) and the data analyzed as described
above. Absolute numbers were determined by multiplying
the mean relative percentages by the mean lymphocyte
number.
Apoptosis assays
Apoptosis was assayed by surface staining for phos-
phatidylserine using Annexin V-FITC (Immunotech,
France) according the manufacturer’s instructions. Samples
were analyzed by flow cytometry by gating on total cells
and analyzing CD3+ lymphocytes for apoptosis. In general,
1.0 � 106 PBMCs/ml from Tg and control rats were
stimulated for 2.5 and 5 h with 25 ng/ml of PMA and 1
Ag/ml of ionomycin (PMA-I), then stained with Annexin
V-FITC and PE anti-rat CD3 (1F4, BD PharMingen). Data
is represented as the difference from stimulated and non-
stimulated lymphocytes in the percent of Annexin V
incorporation.
Statistics
Mean lymphocyte numbers were compared using an
independent Student’s t test for the IFN-g ELISA, total
lymphocyte counts, CD4+, CD8+, and B-lymphocytes
comparisons. The mean percent of CD45RC+ and
CD62L+ subsets, the mean percent of IFN-g producing
CD4+ and CD8+ T cells, and the percent of apoptotic
CD3+ cells were compared within and between groups by
the Wilcoxon rank sum test for samples exhibiting non-
normal distribution. Two-tailed P values were considered
significant at P < 0.05.
21 (2004) 111–119
Acknowledgments
This work was supported by Public Health Service Grant
1K08 AI01792. We thank Odell Jones and Karen Nichols
for their help with the animal studies and Peter O’Driscoll
for his assistance with the statistics.
W. Reid et al. / Virology 321 (2004) 111–119 119
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