B7-H1 is up-regulated in HIV infection and is a novel surrogate marker of disease progression

doi:10.1182/blood-2002-10-3065Prepublished online December 5, 2002;

Haidong Dong, Renato Maserati, Gene M Shearer, Lieping Chen and Mario ClericiDaria Trabattoni, Marina Saresella, Mara Biasin, Adriano Boasso, Luca Piacentini, Pasquale Ferrante, Disease ProgressionB7-H1 is Upregulated in HIV infection and is a Novel Surrogate Marker of

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1

Immunobiology

B7-H1 is Upregulated in HIV infection

and is a Novel Surrogate Marker of Disease Progression.

running head: B7-H1 in HIV infection

Daria Trabattoni, Marina Saresella†, Mara Biasin, Adriano Boasso, Luca Piacentini, Pasquale Ferrante*,

Haidong Dong§, Renato Maserati^, Gene M. Shearer**,

Lieping Chen§ and Mario Clerici.

Chairs of Immunology and *Virology, DISP LITA VIALBA, University of Milano, 20157 Milano; † Dept. of Biology,

Don C. Gnocchi Foundation, IRCCS, 20157 Milano; ** EIB, NCI, NIH, Bethesda 20892, MD, USA § Dept. of

Immunology, Mayo Clinic, Rochester 55095, MN, USA; ^ Infectious Diseases Clinic, IRCCS San Matteo, 29170 Pavia,

Italy.

This paper was supported by grants from Istituto Superiore di Sanita' "III Programma Nazionale di Ricerca sull’ AIDS

1999” and from MURST “Progetto Centri di Eccellenza”.

Correspondence to:

Mario Clerici, M.D. Cattedra di Immunologia, Universita' degli Studi di Milano,

DISP LITA Vialba; Via GB. Grassi, 74, 20157 Milano, ITALY.

Phone: 39.02.5031 9679; Fax: 39.02.5031 9677 e-mail: [email protected]

word count Abstract: 199; Text: 3791

Copyright (c) 2002 American Society of Hematology

Blood First Edition Paper, prepublished online December 5, 2002; DOI 10.1182/blood-2002-10-3065 For personal use only. by guest on May 30, 2013. bloodjournal.hematologylibrary.orgFrom

mailto:[email protected]



2

ABSTRACT

The ligation of programmed death-ligand 1 (B7-H1) to T cells results in the preferential production of IL-10. We

investigated if B7-H1 would be upregulated in HIV infection, a disease characterized by increased IL-10

production, by measuring B7-H1, B7-1 (CD80), and B7-2 (CD86) expression and mRNA in 36 HIV-infected

patients and in 22 healthy controls (HC). Results showed that: 1) B7-H1 expression and mRNA are augmented in

cells of HIV patients; 2) increased IL-10 production in these patients is largely induced by B7-H1-expressing

CD14+ cells; 3) an inverse correlation is detected between B7-H1 expression and CD4 counts, whereas the

upregulation of B7-H1 is directly associated with HIV plasma viremia; 4) antiviral therapy results in the parallel

down modulation of IL-10 production and B7-H1 expression/synthesis; and 5) B7-H1/CD80 and B7-H1/CD86

mRNA ratios are increased in PBMC of HIV patients compared to HC. B7-H1 synthesis and expression is

upregulated in HIV infection and the degree of dysregulation correlates with the severity of disease. Aberrant

antigen presentation by APC that exhibit increased B7-H1 expression and IL-10 production in HIV infection

could be responsible for T lymphocyte unresponsiveness and loss of protective immunity. B7-H1 is a surrogate

marker potentially involved in AIDS disease progression.

[email protected]

Keywords: T lymphocytes, cell mediated immunity, AIDS, costimulation, interleukin-10


mailto:[email protected]



3

INTRODUCTION

The activation of T lymphocytes is dependent on the presentation of processed antigenic peptides in association with

major hystocompatibility molecules (MHC) to lymphocytes that express a T cell receptor specific for that binary

complex (1, 2). However, optimal lymphocyte activation requires a second signal that is delivered by the interaction

between co-stimulatory, accessory molecules (3, 4). The cross-linking of CD28 on the surface of T lymphocytes allows

for the activation of these cells. CD28 binds to a family of ligands on the surface of non-T cells that are collectively

known as B7 molecules (5, 6). Beside B7.1 (CD80) and B7.2 (CD86), a number of other B7-like ligands have been

described (7-9). Among these, B7-H1 is particularly interesting. B7-H1, also called PD-L1, is constitutively present on

monocytes and could be induced on activated T cells. (7). B7-H1 does not interact with CD28, CTLA-4 or ICOS but

was recently shown to bind to PD-1 (10, 11) a CTLA-4- like molecule belonging to immunoglobulin superfamily.

Recent studies, however suggested that receptors other than PD-1 can also ligate B7-H1 (7). Ligation of B7-H1 to T

cells results in the preferential production of IL-10 (7), and in increased T helper dependent synthesis of TNP-specific

IgG2a (12) in mice. These results suggest that ligation of B7-H1 may be responsible for promoting type-2 cytokine-

biased responses.

Interelukin-10 production by peripheral blood mononuclear cells (PBMC) is augmented in infectious and non-infectious

pathologies including HIV disease (13-16). In particular, cell mediated immunity (CMI) is characteristically impaired in

HIV infection (17-20). The progression of this infection is associated with increased HIV replication, reduction of

circulating CD4+ T lymphocytes, functional defects of CMI, and augmented apoptosis of T lymphocytes (21, 22). An

impairment in the production of type 1 cytokines accompanied by increased secretion of type 2 cytokines has also

repeatedly been suggested to accompany progression of HIV disease (14, 16, 23, 24). Because IL-10, a TH2-type

cytokine, is known to be a powerful inhibitor of TH1 activation and to suppress CMI in mice and humans (25, 26), we

studied the possible role of impaired regulation of B7-H1 expression/synthesis in this disease. Furthermore, because

highly active antiretroviral therapy (HAART) was shown to reduce IL-10 production (27-30), we investigated whether

B7-H1 would be down-regulated upon HAART.




4

The present study demonstates that the cell surface expression and the amount of B7-H1 mRNA are increased in HIV–

infected patients, and that those HIV-infected individuals whose disease is immunologically or virologically more

advanced showed an even greater increase in B7-H1. Additionally, we observed a parallel decrease in IL-10 production

and in the expression/synthesis of B7-H1 in HAART-treated patients. Our results suggest that B7-H1 is a surrogate

marker for AIDS disease progression. In addition, our results also implicate a role of B7-H1 in AIDS progression.




5

MATERIALS AND METHODS

Demographic, clinical, and immunological classification of the patients: Immune parameters were investigated in 36

chronically HIV-infected (>3 years of infection) patients. Twenty-four of 36 patients were receiving highly active

antiretroviral therapy (HAART), whereas the other 12 patients were not receiving antiviral drugs. Treated patients had

undergone therapy for a median of 10 months before study period and were antiretroviral-naive prior to starting

HAART. CD4 counts (HAART-treated: mean 589/µl, range 18-1212; HAART-untreated: 507/µl; 309-808) and HIV

viral load (HAART-treated: 2019/ml, <50-33000; HAART-untreated: 8077/ml; 68-45700) were not statistically

different between the two groups of patients. Eighteen patients were treated with one protease inhibitor (PI) plus two

nucleoside reverse transcriptase inhibitors (NRTI); 6 other patients received 2 PI in association with two NRTI. Twenty-

two age-matched HIV-uninfected healthy individuals (blood donors) were studied as well as controls. Written informed

consent was obtained from all the participants and the study was approved by IRBs from all partecipating centers.

Blood samples collection: Whole blood was collected by venopuncture in vacutainer tubes containing EDTA (Becton

Dickinson & Co., Rutherford, NJ). PBMC were separated on lymphocyte separation medium (Organon Teknica Corp.,

Durham, NC), washed twice in Phosphate buffered saline (Organon Teknica) and the number of viable leukocytes was

determined by trypan blue exclusion. All analyses were performed on freshly collected cells.

Stimulation of PBMC: 2x106 PBMC were incubated for 18 hours with 1) medium, 2) PHA (2,5 µg/ml) (Sigma,St.

Louise, MO), or with 3) LPS (10 µg /ml) (Sigma). For cytokine production PBMC were incubated in the

presence/absence of staphylococcal enterotoxin B (SEB, 200ng/ml)(Sigma) + anti CD28 (1µg/ml)(R&D Systems,

Minneapolis, MN).

Immunofluorescent staining: PBMC were washed in PBS, split in different flow cytometry tubes and stained with

monoclonal antibodies specific for CD3, CD14 , CD19, CD80, CD86 (Caltag Laboratories, Inc., Burlingame, CA) for

30 min at 4°C in the dark. For indirect immunofluorescence staining, PBMC were first incubated with with monoclonal

antibody against B7-H1 (7) at 1:1000 dilution. After 30 min at 4°C, the cells were washed and further incubated for 30




6

min at 4°C with goat against human immunoglobulin G F(ab') conjugated with fluorescein isothiocyanante (1:25

dilution)(Caltag Laboratories).

For analysis of cytokine-secreting cells, PBMC were washed after phenotypic staining and fixed in Reagent A solution

(FIX & PERM cell permabilization Kits; Caltag Laboratories, Inc.) for 10 min at room temperature in the dark. The

cells were washed in PBS and resuspended in Reagent B (FIX & PERM cell permabilization Kits; Caltag Laboratories,

Inc.), with cytokine-specific monoclonal antibodies (IL-10 PE). After a 30 min incubation at 4°C in the dark, the cells

were washed and fixed in 1% paraformaldehyde in PBS.

Monoclonal antibodies (mAbs): The following mAbs were used in this study: anti CD3 (mouse IgG2a isotype), anti

CD14 (mouse IgG2a isotype), anti CD19 (mouse IgG1 isotype) coupled to R-phycoerythrin-Cyanine 5 Tandem (TC),

anti CD80 (mouse IgG1 isotype) coupled to R-phycoerythrin (PE), anti CD86 (mouse IgG1 isotype) coupled to

flourescein isothiocyanate (FITC). The intracellular molecule detection mAb used was: anti-human IL-10 (mouse IgG1

isotype) coupled to phycoerythrin (PE).

Cytometric analysis: The cytometric analyses of phenotype and cytokine-secreting lyphocytes were performed using an

EPICS XL flow cytometer (Beckman-Coulter Inc., Miami, FL) equipped with a single 15 mW argon ion laser operating

at 488 nm interfaced with 486 DX2 IBM computer (IBM, UK). For each analysis, 20,000 events were acquired and

gated on CD3 (or CD14) expression, and side scatter properties. Green florescence from FITC (FL1) was collected

through 525-nm bandpass filter, orange-red flourescence from R-PE (FL2) was collected through a 575-nm bandpass

filter, and deep-red fluorescence from TC (FL4) was collected through 670-nm bandpass filter. Data were collected

using linear amplifiers for forward and side scatter and logarithmic amplifiers for FL1, FL2 and FL4. Samples were first

run using isotype control or single fluorochrome-stained preparations for colour compensation.

RNA extraction and reverse transcription: Total RNA was extracted from lymphocytes with the acid guanidium

thiocyanate-phenol-chloroform method and purity was determined by spectrophotometry. RNA was treated with

RNase-free DNase (RQ1 DNase, Promega, Madison, Wisconsin, USA) to remove the contamination of genomic DNA.

One µg of total RNA from lymphocytes was reverse transcribed into first-strand cDNA in a 20 µl final volume




7

containing 1 µM of random hexanucleotide primers, 1 µM of oligo dT and 200 U of Molony murine leukemia virus

reverse transriptase. (Clontech, Palo Alto, CA, USA).

Quantification of CD80, CD86 and B7-H1 cDNA by competitive PCR: To quantify the expression of CD80, CD86 and

B7-H1 we used an exogenous competitor in competitive PCR. The competitor template was built according to the

instruction provided with the Competitive DNA Construction Kit (Takara, Otsu, Japan). The target template and the

competitor have similar lengths and the same primer recognition sequences, thus ensuring identical thermodynamics

and amplification efficiency for both template species. The amount of competitor is known. For CD80 cDNA

quantification the target generates a fragment of 303bp and the competitor is 344bp; for CD86 the target is 403bp, and

the competitor 444bp; for B7-H1 the target is 396bp and the competitor 442bp. After amplification, products are

distinguished by gel electrophoresis to allow densitometric evaluation of the relative intensities of the bands. The ratio

of amplification products reflects the ratio between the initial amount of template, thus allowing the precise evaluation

of CD80, CD86 and B7-H1 cDNA amounts. The same procedure has been followed to quantify the GAPDH cDNA

(target 199bp; competitor 241bp), in order to normalize the results obtained. The magnitude of target gene expression is

then calculated as copies of target cDNA per copies of GAPDH cDNA.

Quantification of cytokine cDNA by PCR: To normalize the cDNA sample concentration, all samples were diluited to

the same concentration as the sample with the lowest GAPDH concentration. Each PCR was performed in a 25 µl of

reaction mixture containing 5 µl of diluited RT reaction mixture, 2.5 µl of 1X PCR buffer (20 mM Tris-HCl, 100 mM

KCl, 0,1 mM EDTA, 1 mM DTT, 0,5% Tween 20, 0,5% Nonited p40, 50% Glycerol), 1.5 µl of 25 mM MgCl2, 2 µl of

200 µM concentration of each dNTP, 0,2 µl of 1,25 Units of Taq polymerase (Takara, Otsu, Japan), 1 µl of 0,4 µM

GAPDH primers and 1 µl of 0,4 µM for each cytokine (IL-2, IL-10). Thermal cycling was performed in a Touchdown

Hybaid (Celbio, Milano, Italy) by using the following amplification profile: an initial denaturation at 95°C for 10 min;

35 cycles of denaturation at 95°C 30 sec, annealing at 60°C 30 sec, and extension at 72°C 30 sec; a final amplification

step at 72°C 10 min.

The PCR reaction products were then electrophoresed in a 10% acrylamide gel and stained with 0,5 µg/ml ethidium

bromide, and the size of each cDNA product was determined by comparison to a DNA size marker pbR322 (Sigma, St.

Louis, MO, USA). To quantify relative levels of gene expression the bands on the gels were scanned by transmission




8

densitometry, and the areas of the peaks were calculated in arbitrary units. To evaluate the relative levels of expression

of the target genes in RT-PCRs, the value of the internal standard (GAPDH) in each test tube was used as the baseline

gene expression of that sample, and the relative value was calculated for each of the target genes amplified in that

reaction. These values were then used to compare expression across samples tested.

Statistical analysis: Procedures were based on non parametric analyses (Mann-Whitney); comparisons between

different groups of patients were made using Fisher’s exact 2-tailed test. Statistical analysis was performed using the

SPSS-PC statistical package (SPSS inc. Chicago, IL).




9

RESULTS

IL-10 production is increased in HIV patients:

IL-10 and IL-2 mRNA were quantified in unstimulated and in anti CD3+ anti CD28-stimulated PBMC of 36 HIV-

infected individuals and 22 HC. IL-10-specific mRNA was increased and IL-2-specific mRNA was reduced in HIV

patients compared to HC. These differences were statistically significant and were observed both in ustimulated and in

antiCD3+antiCD28-stimulated PBMC (Figure 1).

B7-H1-expressing CD3+, CD14+ and CD19+ cells of HIV infected individuals and healthy controls:

B7-H1 is expressed on resting monocytes/macrophages and B lymphocytes and appears on activated T cells. Thus, we

measured B7-H1 expression by FACS in unstimulated and in mitogen-stimulated CD3+, CD14+ and CD19+ cells from

all the HIV-infected individuals and HC included in the study. PBMC were either stimulated with PHA and

subsequently gated on CD3, or stimulated with LPS and gated on CD14 or CD19. Results are shown in Figure 2 and are

summarized as follows: 1) the percentage of unstimulated and mitogen-stimulated CD3+/B7-H1+ cells was comparable

in HIV-patients and controls; 2) B7-H1-expressing unstimulated and mitogen-stimulated CD14+ cells were

significantly increased in HIV-patients compared to controls (p= 0.024 and p= 0.011, respectively); 3) unstimulated and

mitogen-stimulated CD19+/B7-H1+ cells were significantly increased in HIV-patients compared to controls (p= 0.014

and p= 0.0012, respectively). Representative results obtained in HIV-infected patients and in healthy controls are shown

in Figure 3.

The above results showed that HIV infection is associated with the upregulation of B7-H1, and ligation of B7-H1 is

known to result in the preferential generation of IL-10. To verify whether all the cell types analyzed produce increased

amounts of this cytokine, we analyzed intracellular IL-10 production in CD3+ (distinguishing between CD4+ and CD8+

lymphocytes), CD14+, and CD19+ B7-H1-expressing cells of HIV-infected patients and HC. The results showed that,

although all B7-H1-expressing cell types stained positive to intracellular IL-10, increased IL-10 production in HIV

infection was mainly observed in the B7-H1-expressing CD14+ cells (Fig. 4).




10

B7-H1-specific mRNA in PBMC of HIV infected individuals and healthy controls

B7-H1-specific mRNA was quantified using PCR techniques in unstimulated and anti CD3+anti CD28-stimulated

PBMC of HIV-infected patients and HC. The results indicated that PBMC of HIV-infected patients expressed a

significantly higher amount of B7-H1-specific mRNA compared to HC. These differences were statistically significant

both when unstimulated (p= 0. 024) and anti CD3+anti CD28-stimulated PBMC (p= 0. 032) were analyzed. CD80- and

CD86-specific mRNA was quantified as well; results showed that similar amounts of CD80 and CD86-specific mRNA

are present in unstimulated and mitogen-stimulated PBMC of patients and controls (Table 1). Representative results

obtained in HIV patients and in healthy controls are shown in Fig. 5.

Correlation of B7-H1 expression with CD4 count and HIV viral load in HIV-infected patients

The progression of HIV infection is associated with reduced CD4 counts, increased HIV plasma viremia, and increased

IL-10 production. To analyze possible associations between these parameters we tested whether B7-H1 expression

would be correlated with CD4 counts and HIV viremia. A number of statistically significant correlations were observed.

Thus, results indicated an inverse correlation between CD4 counts and the percentage of unstimulated CD3+/B7-H1 (p=

0. 032; r= -0. 396)(Fig. 6, panel A) and CD19+/B7-H1 (p= 0. 012; r= -0. 426)(Fig. 6, panel B) cells. An inverse

correlation was also seen between CD4 counts and the percentage of mitogen-stimulated CD14+/B7-H1+ (p= 0. 028; r=

-0.381)(Fig. 6, panel C) and CD19+/B7-H1+ (p= 0. 011; r= -0.521)(Fig. 6, panel D) cells. These results were verified

by quantification of B7-H1-specific mRNA. Results confirmed that CD4 counts are inversely correlated with the

expression of B7-H1-specific mRNA in unstimulated PBMC (p= 0. 0045; r= -0.620)(Fig 6, panel E).

The potential value of B7-H1 as a surrogate marker of progression in HIV infection was further reinforced by the

positive correlation that was detected between HIV plasma viremia and the percentage of unstimulated (p= 0. 012; r=

0.461)(Fig. 6, panel F) and mitogen-stimulated (p= 0. 032; r= 0.377)(Fig. 6, panel G) CD19+/B7-H1+ cells. Finally, a

strong positive correlation was detected between HIV plasma viremia and IL-10 producing, B7-H1-expressing CD14+

cells (p= 0. 008; r= 0.819).




11

Antiviral therapy reduces IL-10 production, resulting in a down-regulation of B7-H1

Highly active antiretroviral therapy (HAART) results in a reduction of IL-10 production. We verified if this effect of

HAART would be associated with a down regulation of B7-H1. Thus, we divided HIV-infected individuals into those

who were (n= 24) or were not (n= 12) undergoing HAART, and compared B7-H1 expression and B7-H1-specific

mRNA in the two groups of patients. CD4 counts and HIV viral load were not statistically dissimilar between these

groups of individuals.

The results showed that IL-10 was reduced in HAART-treated patients; this result was paralleled by a down-regulation

of B7-H1. Thus: 1) the percentage of unstimulated and of mitogen-stimulated peripheral cells is reduced in HAART-

treated patients; and 2) B7-H1-specific mRNA is reduced in unstimulated and mitogen-stimulated PBMC of HAART-

treateed compared to HAART-untreated patients. These results are shown in Table 2




12

DISCUSSION

T cell activation is induced by the presentation of processed antigenic peptides within the cleft of MHC molecules on

the surface of antigen presenting cells (APC) in the presence of cytokines and costimulatory molecules (1-4). The B7

family of molecules is the main cluster of costimulatory molecules expressed on the surface of APC and include both

molecules with a prevalent stimulatory effect (CD80 and CD86) and molecules that can have a prevalent inhibitory

function (6, 7, 31). Among these molecules B7-H1, a receptor that is mainly present on monocytes and on B

lymphocytes but also appears on T lymphocytes upon activation, has a pivotal role (7). B7-H1 does not interact with

CD28, CTLA-4 or ICOS and was recently demonstrated to ligate PD-1 (10). Nevertheless, because PD-1 is only

detected on a minority of activated T cells (7), it has been suggested that receptors other that PD-1 can also ligate B7-

H1. Ligation of B7-H1 to its receptor(s) results in the preferential stimulation of IL-10 production, and in the synthesis

of T helper dependent TNP-specific IgG2a in mice (7, 8). Thus, ligation of B7-H1 could be responsible for promoting

type-2 cytokine-biased responses. Antigen presentation in the presence of IL-10 does not lead to T cell proliferation but

rather to lack thereof (25, 26), ligation of B7-H1 may therefore also result in the unresponsiveness/anergy of antigen-

specific T cells. Recent results show that ligation of B7-H1 also promotes T cell apoptosis, potentially favoring immune

evasion of tumors (31).

HIV infection is associated with increased IL-10 production (13-16) and is also associated with functional impairment

and anergy of antigen-specific responses, and increase susceptibility of T lymphocytes to apoptosis (21, 22). These

clinical observations are strikingly similar to the effect induced by B7-H1 ligation. Thus, we evaluated whether the

synthesis and expression of B7-H1 would be upregulated in HIV infection and in progression to AIDS. Our results

demonstrated that B7-H1 is upregulated in HIV infection and that the degree of upregulation is negatively associated

with CD4 counts and positively associated with HIV plasma viremia. Furthermore, parellel decreases in IL-10

production and in B7-H1 synthesis/expression are seen during antiretroviral treatment of HIV-infected patients. Our

observation supports B7-H1 expression as a reverse indicator for HIV infection.




13

It is unknown whether B7-H1 is also involved in the downregulation of CMI which is observed in HIV infection. The

observation that B7-H1 ligation preferentially stimulates secretion of IL-10 (7), which downregulates Th1 response and

facilitates the induction of T cell anergy (25, 26), seems to support this hypothesis. The mechanism(s) leading to the

upregulation of B7-H1 in HIV infection is unclear but this phenomenon is unlikely to be secondary to a direct effect of

the virus. In fact, recent data show that B7-H1 is abundant in human carcinomas of lung, ovary, and colon (32), whereas

the synthesis and expression of B7-H1 is drastically reduced in inflammatory bowel diseases (IBD) (Clerici et al.,

manuscript in preparation). Interestingly, whereas carcimomas (33-35) are known to be associated with increased IL-10

production, IL-10 production is reduced in IBD (36-38).

HIV infection results in a loss of T helper function which involves all the T helper/APC pathways. In particular, the loss

of T helper cell proliferation and IL-2 production in response to presentation of processed antigens on self APC is

precocious and characteristic (18). Earlier data stemming from co-coltures of HIV-infected T helper cells with

autologous uninfected APC, or vice versa, showed that this defect is not dependent either on an inability of the APC to

process/present antigens, or on a T helper cell-specific impairment, but rather on the way in which the two cell types

interact (39, 40). The observation that B7-H1 expression is augmented on the surface of cells of HIV-infected

individuals could explain this observation. Thus, antigenic presentation in the presence of IL-10 secretion could results

in a lack of T cell responsiveness.

The susceptibility of antigen-stimulated CD4+ T cells of HIV-infected patients to undergo apoptosis is enhanced (41-

43), and vitro susceptibility of these lymphocytes to apoptosis was shown to be differentially regulated by type 1 and

type 2 cytokines. Hence, type 1 cytokines prevent in vitro apoptosis, whereas type 2 cytokines, and in particular IL-10,

did not prevent, but amplified apoptosis (44, 45). Ligation of B7-H1 on APC of HIV-infected individuals, and

subsequent IL-10 production, would thus result in augmented susceptibility of antigen-specific T cells to apoptosis. It is

interesting that the inhibitory effects associated with the triggering of the PD-l pathway, and the prevention of IL-10-

induced apoptosis of CD4+ T lymphocytes of HIV-infected individuals, can be overcome by IL-2 (45). Nevertheless,

this rescue mechanism would likely not function in progressive HIV infection, a disease which is characterized by early

defects that selectively involve IL-2 production (17, 18).




14

We demonstrate here a direct correlation between HIV viral load and: 1) increased expression of B7-H1, and 2)

augmented percentage of B7-H1-expressing, CD14+, IL-10+ cells. It has been recently reported that IL-10 enhances

entry of HIV into target cells through the upregulation of CD4 and CCR5 (46, 47). Additionally, IL-10 was shown to

increase HIV infection of human monocytes (48) and to directly stimulate viral replication in antigen presenting cells

(49). The B7-H1-mediated augmented production of IL-10 could explaine these observations.

Although HIV infection resulted in increased B7-H1 synthesis/expression on CD3+, CD14+, and CD19+ cells, our data

show that IL-10 production in HIV infection is mostly observed in CD14+ cells. This result confirms previous data

demonstrating that IL-10 production is differentially regulated in T cells and monocytes of HIV-infected individuals,

and that monocytes are the major IL-10-producing cell type in this infection (50). The potential of IL-10 producing-

antigen presenting cells to modulate immune responses has been reported in experimental autoimmune encephalitis

(EAE) in mice (51, 52). In this model, suppression of EAE is dependent on the ability of APC to produce IL-10 (51,

52), and IL-10 production can result in tolerance via a direct suppression of T helper lymphocytes (53), via the

modulation of regulatory cells (54), or through the modulation of APC function (55). The importance of the the PD-l

pathway in the regulation of tolerance, or lack thereof, is further reinforced by results showing that mice deficient in

PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features (11, 56, 57). In this

context, it is noteworthy that progressive HIV disease is associated with autoantibody production (58, 59). The

dysregulation of the PD-l pathway resulting in the upregulation of synthesis/expression of B7-H1 in the context of a

chronic infection, as described here in HIV infection, could nevertheless be deleterious and lead to tolerance and

unresponsiveness of antigen-specific lymphocytes, phenomenona that are seen in this infection. In this time, however,

we do not know whether the effect of B7-H1 is mediated through ligation of PD-1 on T cells because other receptors

other than PD-1 on T cells have been implicated in the effect of B7-H1.

Biological phenomena that are associated with progression of HIV-infected individuals to AIDS are: 1) decline in the

number of CD4+ T cells; 2) increase in HIV plasma viremia; 3) impairment/anergy of the functionality of T helper

cells, decrease in production type 1 cytokines, and increase in IL-10 production; and 4) increased susceptibility of




15

PBMC to activation-induced T cell death. This study sheds light on the possible connection among these phenomena by

demonstrating that B7-H1 synthesis/expression, the ligation of which results in IL-10 production, T helper cell

impairment/anergy , and promotion of T cell apoptosis is augmented in the progression of HIV infection. Blockade of

B7-H1 by specific antibodies or soluble inhibitors could be beneficial in HIV disease.




16

ACKNOWLEDGMENTS

We are grateful to Ms. Alessandra Beardo, Giuliana Magri, and Francesca Fasano, Department of Immunology,

University of Milano, for excellent technical support.




17

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Table I B7-H1-, CD80-, and CD86- specific mRNA in unstimulated and in αCD3+αCD28-stimulated PBMC of healthy

controls and of HIV infected patients. Protein/GAPDH ratio is presented. Mean values and S.E. are shown.

Unstimulated anti CD3+ antiCD28-stimulated _

B7-H1 CD80 CD86 B7-H1 CD80 CD86

Healty controls (N= 22) 24.3 + 6.8* 29.2 + 8.3 44.7 + 14.1 74.1 + 14.2** 57.1 + 3.6 83.5 + 22.4

HIV-patients (N=36) 91.7 + 17.3 38.9 + 7.4 25.2 + 5.8 151.8 + 18.6 72.1 + 8.9 43.6 + 9.7

* p< 0. 05

** p< 0. 005




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Table II. IL-2 and IL-10 mRNA, B7-H1 expression, and B7-H1-specific mRNA in 24 HIV-infected

patients undergoing highly active antiretroviral therapy (HAART) and in 12 HIV-

infected, HAART-untreated patients. Mean + S.E. and statiscal significance are shown.

HIV-infected individuals____________________

HAART-treated HAART-untreated p value

Unstimulated PBMC

IL-2 mRNA 0.53 + 0.31 0.88 + 0.29 n.s.

IL-10 mRNA 3.06 + 1.02 5.59 + 0.97 n.s.

B7-H1 expression on:

CD3+ cells 5.34 + 1.44 8.98 + 2.51 n.s.

CD14+ cells 19.37 + 4.88 33.71 + 8.55 0. 043

CD19+ cells 26.74 + 4.58 39.91 + 7 .15 0. 032

B7-H1-specific mRNA 19.04 + 4.17 31.14 + 5.88 0. 033

Stimulated PBMC

IL-2 mRNA 2.06 + 0.87 3.18 + 0.99 n.s.

IL-10 mRNA 3.62 + 0.95 6.72 + 1.12 0. 017

PDL-1 expression on:

CD3+ cells 9.98 + 2.54 14.17 + 3.21 n.s.

CD14+ cells 27.44 + 4.19 39.81 + 5.72 0. 039

CD19+ cells 37.81 + 4.77 46.11 + 6.22 n.s.

B7-H1-specific mRNA 78.12 + 9.15 107.74 + 14.32 0. 041




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FIGURE LEGENDS:

Figure 1. IL-12 and IL-10-specific mRNA in unstimulated (panel A) and anti CD3+ anti CD28-stimulated PBMC of

HIV-infected individuals (black box) and of healthy controls (white box). Mean values, S.E., and statistically significant

differences are shown. *= p< 0. 05; **= p< 0. 005.

Figure 2. Percentage of B7-H1-expressing CD3+, CD14+, and CD19+ cells of HIV-infected individuals (black box)

and of healthy controls (white box). Cells were either unstimulated (panel A) or mitogen-stimulated. Mean values, S.E.,

and statistically significant differences are shown. *= p< 0. 05; **= p< 0. 005.

Figure 3A. Flow cytometry analysis of B7-H1 expression by CD3+, CD14+, and CD19+ unstimulated cells of a

representative healthy control (upper panels) and a representative HIV-infected patients (lower panels).

Figure 3B. Flow cytometry analysis of B7-H1 expression on CD3+, CD14+, and CD19+ anti CD3+ anti CD28-

stimulated cells of a representative healthy control (upper panels) and a representative HIV-infected patients (lower

panels).

Figure 4. Flow cytometry analysis of intracellular IL-10 in CD4+, CD8+, CD14+ and CD19+ unstimulated cells of a

representative healthy control (upper panels) and a representative HIV-infected patients (lower panels).

Figure 5. Upper panels. Quantification of B7-H1-specific mRNA in resting (left panel) or in antiCD3+antiCD28--

stimulated (right panel) PBMC of three representative healthy controls.

Lower panels. Quantification of B7-H1-specic mRNA in resting (left panel) or in anti CD3+ anti CD28-stimulated

(right panel) PBMC of three representative HIV-infected patients. The upper bands show cDNA retrotranscribed from

mRNA extracted from PBMC; the lower bands show diferent dilutions of the specific competitors. The arrows show the

dilution at wich equivalence is observed between sample cDNA and the specific competitors.




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Figure 6. Correlation between B7-H1 expression and CD4+ T cells counts (panels A, B, C, D) and between B7-H1

expression and HIV plasma viremia (panels E, F) in HIV-infected patients. Upper panels show the correlation between

CD4 counts and the percentage of: unstimulated CD3+/B7-H1 (A); unstimulated CD19+/B7-H1 (B); mitogen-

stimulated CD14+/B7-H1 (C); and mitogen-stimulated CD19+/B7-H1 (D). The middle panel shows the correlation

between CD4 counts and the expression of B7-H1-specific mRNA in unstimulated PBMC (E). Lower panels show the

correlation between HIV plasma viremia and the precentage of unstimulated (F) and mitogen-stimulated CD19+/B7-H1

(G) cells




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B7-H1 is up-regulated in HIV infection and is a novel surrogate marker of disease progression

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