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: reid@umbi.umd.edu (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

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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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