J Med Primatol 2000: 29: 1–10Printed in Ireland - all rights reser6ed.

Study of immunological and virologicalparameters during thalidomide treatment ofSIV-infected cynomolgus monkeys

Di Fabio S, Trabattoni D, Geraci A, Ruzzante S, Panzini G, Fusi ML,Chiarotti F, Corrias F, Belli R, Verani P, Dalgleish A, Clerici M, TittiF. Study of immunological and virological parameters during thalido-mide treatment of SIV-infected cynomolgus monkeys. J Med Primatol2000; 29:1–10. © Munksgaard, Copenhagen

Abstract: The potential therapeutic utility of thalidomide (Thd), aneffective inhibitor of tumor necrosis factor (TNF)-a in 6itro, was inves-tigated in cynomolgus monkeys (Macaca fascicularis) at 10 monthsafter infection with simian immunodeficiency virus (SIV). Thd-treatedmacaques (n=8) received an oral dose (10 mg) daily for 7 days, fol-lowed by a wash-out period of 5 weeks. A 2nd cycle of treatment wasperformed on the same animals at higher doses (20 mg Thd/day) for 14days. The control monkeys (n=7) received a placebo for the sameperiod of time. In the present study, we show that Thd, in addition toinhibiting TNF-a production after in 6itro mitogen stimulation of pe-ripheral blood mononuclear cells (PBMCs), was able to restore theproliferative responses to SIV peptides in monkeys that were infectedwith SIV. Interestingly, we found that such effects are associated withan increased expression of CD28 cell surface receptors on CD4+ T-cells paralleled by a decrease on CD8+ T-cells. At the same time,significant reduction in either cell-associated viral load or plasma viralRNA was not observed among the SIV-infected monkeys during thetwo treatment cycles, when compared with the placebo group.

S. Di Fabio1, D. Trabattoni2,A. Geraci1, S. Ruzzante2,G. Panzini1, M.L. Fusi2,F. Chiarotti3, F. Corrias1,R. Belli1, P. Verani1,A. Dalgleish4, M. Clerici2,F. Titti11Laboratory of Virology, Istituto Superiore diSanita, Rome, 2Department of Immunology,University of Milan, Division L.I.T.A., L.Sacco Hospital, Milan, 3Laboratory ofOrgan and System Pathophysiology, IstitutoSuperiore di Sanita, Rome, Italy, 4Divisionof Oncology, St. George’s Hospital MedicalSchool, University of London, London,United Kingdom

Key words: CD28 – proliferative responses– SIV – thalidomide – TNF-a

Accepted July 19, 1999.

Dr. F. Titti, Laboratory of Virology, IstitutoSuperiore di Sanita, Viale Regina Elena,299, 00161 Rome, Italy.E-mail: titti@virus1.net.iss.it

Introduction

Infection with human immunodeficiency virus(HIV) in humans or with simian immunodefi-ciency virus (SIV) in non-human primates is asso-ciated with a complex alteration of the cytokinenetwork that could have a pathologic role in theprogression of HIV/SIV infection to acquired im-munodeficiency syndrome (AIDS) [3, 5]. Tumornecrosis factor (TNF)-a, a pleiotropic cytokineproduced primarily by monocytes andmacrophages, has been reported to enhance HIVreplication via the activation of the transcriptionalfactor NF-kappa B upon binding the long termi-nal repeat (LTR) sequences of HIV [12]. Increas-ing concentrations of this cytokine observedduring the progression of the disease have beenassociated with the onset of opportunistic infec-tions and with pathological status including fever,weakness, tissue necrosis and severe weight loss

[23]. However, other reports indicated that TNF-ain 6itro inhibits the replication of both deoxyri-bonucleic acid (DNA) and ribonucleic acid(RNA) viruses [28, 40]. Therefore, drug therapyaimed at reducing TNF-a production has beenproposed as an effective defense against HIV in-fection [20]. Pentoxifylline, a trisubstituted xantinederivative, has been reported to improve cell-me-diated immunity and to reduce plasmaviremia inasymptomatic patients infected with HIV. How-ever, the effect of pentoxifylline on the regulationof TNF-a expression is controversial [4, 11].Thalidomide (Thd) is a drug known to suppressTNF-a production by human monocytes in 6itro[36] and its therapeutic use has been shown tohave beneficial, although limited, effects on a vari-ety of clinical symptoms including HIV infection[15, 21, 32, 38]. Nevertheless, because of itsknown teratogenic effects the use of Thd on hu-mans is limited.

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Di Fabio et al.

To study the effects of Thd in the monkey modelof HIV infection, SIV-infected cynomolgus mon-keys were treated with the drug. Changes in viro-logical (cell-associated viral load and plasmaviremia) and immunological (cell surface markerexpression) parameters, proliferation induced bySIV peptides and mitogen-stimulated TNF-a pro-duction were examined and compared with thoseobtained from placebo-treated SIV-infectedmacaques.

Whereas no clear-cut effect of Thd treatment onvirological parameters could be detected, periph-eral blood mononuclear cells (PBMCs) of Thd-treated macaques showed in 6itro a reduction ofTNF-a production and an improvement in theproliferative responses to SIV peptides that areassociated with an increased expression of CD28on CD4+ and a decreased expression on CD8+

T-lymphocytes. Our results suggest that Thd is amore complex immunomodulatory molecule thanwas previously reported.

Materials and methodsAnimals

Adult male cynomolgus monkeys (Macaca fascicu-laris) used for this study were housed in singlecages within level three biosafety facilities accord-ing to the European guidelines for non-humanprimate care (EEC, Directive No. 86-609, Novem-ber 24, 1986). Blood samples were obtained fromthe inguinal vein while the animals were underketamine hydrochloride anesthesia (10 mg/kg).They were used for hematological analysis and forimmunological and virological assays, which wereperformed in two different laboratories in ablinded fashion. Clinical examination, weight andrectal temperature data were recorded and bloodwas collected after slightly sedating the monkeys.The blood was collected in ethylenediaminete-traacetic acid (EDTA) and plasma was obtainedafter centrifugation at 1,800 g. All samples weretaken between 09:00 and 11:00 hours and all analy-ses were performed on the same day of bleeding.Data on white blood cell (WBC) and red blood cell(RBC) counts, hemoglobin (HGB), hematocrit(HCT), mean corpuscular volume (MCV), meancell hemoglobin (MCH) and mean cell hemoglobinconcentration (MCHC) were obtained using anautomatic particle counter (Datacell 8, Hycel,France). Reticulocytes were counted by examina-tion of slides after staining with Brilliant CresilBleu (Sigma Chemical Co., St. Louis, MO), anddifferential WBCs were counted by examination ofGiemsa May Grunwald (Merck, Darmstadt, Gera-many) stained smears [2].

Treatment and dosing schedule

The animals enrolled in this study were intra-venously infected with 40 MID50 of SIVmac251/32H 10 months before starting the treatment. Atotal of 15 animals were chosen at random anddivided into two groups. In the first group, eightmonkeys received a single oral dose of Thd (10mg/day) resuspended in fruit juice (4 ml) daily for7 days. After a wash-out period of 5 weeks, a 2ndcycle of treatment was performed for the sameanimals for a period of 14 days at higher doses of20 mg/day (resuspended in 6 ml of fruit juice). Theremaining seven monkeys received 4–6 ml of fruitjuice only. Blood samples (10 ml at each bleedingtime) were collected at the beginning and at theend of each cycle of treatment: Days 0 and 7 (t0

and t7) for the 1st cycle and days 0 and 14 (t01 andt14) for the 2nd cycle. In addition, blood sampleswere also collected 7 weeks after the end of the 2ndcycle of therapy.

Cell-associated viral load

Ficoll-Paque (Pharmacia, Uppsala, Sweden)purified monkey PBMCs were isolated from 5 mlof citrated blood. Serial 2-fold dilutions of PBMCsin duplicate (from 1×106 to 4.8×102) were co-cultured in Roswell Park Memorial Institute(RPMI) 1640 medium containing 10% fetal calfserum and antibiotics in 96-well tissue cultureplates (final volume 200 ml) with human CEMXl74cells (1×104; obtained from AIDS Research andReference Reagent Program, Division of AIDS,NIAID, NIH, USA) [35]. The co-cultures wereincubated for 14 days at 37°C, 5% CO2, being fedwith fresh culture medium on days +3, +7 and+10. The presence of SIV-infected PBMCs wasvisually screened every day for syncytia formation.The 50% end point was calculated using themethod of Reed and Muench [34], and the resultswere expressed as the number of infected cells per106 PBMCs.

Viral RNA and p27 determination in plasma

Measurement of SIV p27 gag protein was per-formed in plasma, after acidic antigen–antibodycomplex dissociation using an antigen-capture en-zyme-linked immunosorbent assay (ELISA) test(SIV p27 Core Antigen; Coulter, Hialeah, FL) witha limit of detection of 50 pg/ml. SIV-RNA deter-mination was performed on frozen (−70°C)plasma samples, which were thawed and assayed atthe same time. RNA was extracted from 250 ml ofplasma using the RNA fast isolation system(Molecular Systems, San Diego, CA). Total RNA

2

Thalidomide therapy during SIV infection

(1 mg) underwent reverse transcription and wasamplified by polymerase chain reaction (PCR) us-ing primers specific for the gag region of SIV-mac251 (SG1096Ngag ; SG1592Cgag) in 100 ml ofreaction mixture for a total of 40 cycles in athermal cycler (Perkin Elmer Cetus Co., Emerville,CA), as previously described [39]. The 496 basepair (bp) amplified PCR products were analyzedusing 1.8% agarose gel electrophoresis and visual-ized under ultraviolet (UV) after staining withethidium bromide. Hybridization was achieved at42°C with a 32P-labeled oligonucleotide probe(SG5) and the filters were exposed to X-ray for12–18 hours. The film was scanned (wavelength633 nm) using ULTRO Scan™ XL laser Densito-meter (Pharmacia LKB Biotechnology, Uppsala,Sweden), with a range of 0.01–4 absorbency units(AU). The results of SIV-RNA levels in plasma foreach animal are expressed as the intensity of theAU of the band.

Antigen-stimulated proliferation

Whole blood was diluted 1:10 with phosphate-buffered saline (PBS) and PBMCs were isolated bydensity gradient centrifugation for 40 minutes at800 g using lymphocyte separation medium(Organon Teknika Corp., Durham, NC). PBMCs(3×105) were placed in flat-bottom wells of amicrotitre culture plate (Costar, Cambridge, MA)in a final volume of 200 ml along with: (A) nostimulation (medium background); (B) influenza(Flu) A virus (A/Bangkok RX73 H3N2) (1:500concentration) serving as non-related antigen; or(C) a pool of eight different synthetic SIV peptides(en6 108–122, CITMKCNKSETDKWG; en6 306–320 KY YNLTMKCRRPGNK; en6 430–445RNYVPCHIRQIINTWH; en6 499–511 KLVEIT-PIGL APT; nef 125–138 EKGGLEGIYYSERR;nef 164–177 GPRYPKTFGGWLWK; nef 201–215 SQWDDPWGEVLVWKF; gag 179–190EGCTPYDINQML) (kind gift from Dr. CChougnet, EIB, NCI, NIH, Bethesda, MD) at afinal concentration of 5 mM. Three replicate cul-tures were performed for each stimulation. Pooledmonkey plasma was added to each well (1:20 final)1 hour after sensitization of the PBMCs. For theproliferative assay, cultures were pulsed with 1mCi/well of [3H] thymidine 5 days after antigenicstimulation and harvested 18 hours later. Stimu-lated samples showing a mean cycles per minute(cpm) value higher than the mean cpm value(329.569123) plus 5-fold the standard deviation(SD) of the unstimulated samples were scored aspositive.

Mitogen-stimulated TNF-a production

TNF-a production by PBMCs was determined byculturing 3×106/well PBMCs in 24-well LINBROplates (Flow Laboratories, Inc., McLean, VA) at37°C in a moist, 5% CO2 atmosphere. PBMCswere either unstimulated or stimulated with phty-ohematoglutinin (PHA) (M form, Grand Island,NY) diluted 1:100. These stimuli were chosen be-cause of our experience in measuring PHA-stimu-lated cytokine production and to allow thecomparison of these results with those obtained inprevious studies. The cultures were supplementedwith 5% pooled monkey plasma. Supernatantswere harvested after 48 hours of culture, frozenand stored at −80°C until they were assayed forcytokine production. Cytokine production wasevaluated with a commercially available ELISAassays (TNF-a Predicta, Bender MedSystem, Vi-enna, Austria) with a limit of detection of 1.5pg/ml. Values were calculated from a standardcurve of the corresponding recombinant humancytokine according to the manufacturer’sinstructions.

Lymphocyte subsets determination

Citrated whole blood (100 ml) was used for stainingfor either two-color (B- and T-cell markers) orthree-color (T-cell subsets) analysis using the fol-lowing combination of monoclonal antibodies:Anti-CD20-PE and anti-CD2-FITC (clone L26and clone MT910; Dako A/S Glostrup, Denmark);anti-CD8-PerCP, (Leu-2a; Becton Dickinson Im-munocytometry System, San Jose, CA) combinedwith anti-CD4-PE (clone MT310; Dako A/SGlostrup, Denmark) and anti-CD28-FITC (cloneCD28.2; Immunotech S.A., Marseille, France).Ten thousand lymphocytes of each sample, gatedfrom leukocyte types based on forward and lightscatter, were analyzed using FACScan (BectonDickinson Immunocytometry System, San Jose,CA). Isotype-matched murine immunoglobulinsconjugated to the distinct fluorochromes (BectonDickinson) were used as a control.

Statistical analysis

Data were analyzed using non-parametric meth-ods. In order to verify the statistical significance ofdifferences observed between Thd- and placebo-treated monkeys, three different models were ap-plied, considering the results at (i) t0 and t7, (ii) t01

and t14, and (iii) t0 and t14. In particular, the maineffect of treatment was assessed by applying theMann–Whitney U-test to the mean values of the

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Di Fabio et al.

parameters [(i)–(iii)]; the main effect of time wasassessed by applying the Wilcoxon test to pairedobservations and the interaction effects were evalu-ated using the Mann–Whitney U-test applied tothe differences between paired observations. Multi-ple comparisons were performed using theWilcoxon test with Bonferroni’s correction.

ResultsEvaluation of virological parameters before and after Thdtreatment

The status of the monkeys is summarized in Table1. Following infection with SIVmac 251/32H, allmonkeys seroconverted and became infected asindicated by the frequent isolation of virus fromPBMCs co-cultured with human CEMX174 cells.The 15 SIV-infected animals enrolled for this studyat 10 months after infection were tested for SIVp27 antigen in plasma and for cell-associated viralload in PBMCs before treatment and at the end ofeach cycle. The SIV p27 protein was not detectablein the plasma of all animals on day 0 and duringthe course of the experiment (data not shown).After 7 or 14 days of Thd treatment, no significantreduction of the cell-associated viral load was ob-served in the PBMCs of the treated animals whencompared with those of the animals receiving

placebo (Table 2). We further evaluated the pres-ence of SIV-RNA in the plasma of monkeys beforeand after the 1st cycle of Thd treatment. Eachanimal showed a different pattern for the intensityof the band corresponding to the PCR-amplifiedgag product. No differences on the intensity of theband on day 0 and after 7 days of Thd treatmentwere observed as judged by densitometric analysis(Table 2). All together, these results suggest thatneither the 1st cycle of Thd treatment (7 days) northe longer cycle (14 days) was able to induce adetectable reduction of either the cell-associatedviral load or, at least after the 1st cycle, of viralRNA in plasma of SIV-infected macaques (Table2).

Antigen-stimulated proliferation

Before and after the two cycles of treatment,PBMCs of all monkeys were tested for their capa-bility to proliferate after antigenic stimulation (Fig.1). None of the monkeys in either group wereresponsive to either Flu or SIV peptides before the1st cycle of Thd treatment. After this cycle, apositive Flu response was present in one (14%) ofthe seven monkeys in the placebo group and infour (50%) of the eight Thd-treated monkeys (datanot shown). When SIV peptide-induced prolifera-

Table 1. Summary of the virological and the immunological parameters of the SIV-infected monkeys before Thd treatment

CD4+ T-cells

Day of Thd treatment4Day of infection

Monkey code Strain of virus1 Absolute counts%Absolute counts5%Virus isolation3Anti-SIV Ab2

30.0 1,880 21.615 1,200SIVmac/32H + ++ + 41.9 2,520 39.5 2,080SIVmac/32H16+ + 39.5 3,590 35.0 1,390SIVmac/32H18

1,78025.71,67024.1++SIVmac/32H23+ + 33.4 2,240 15.7 850SIVmac/32H27

36 SIVmac/32H + + 37.0 4,000 30.0 1,4101,13024.62,02043 33.6++SIVmac/32H

44 SIVmac/32H + + 31.4 2,430 37.0 2,080

14 + 40.1 4,910 38.6 2,640SIVmac/32H +20 SIVmac/32H + + 27.0 1,710 9.82 26033 SIVmac/32H + + 38.0 2,320 30.0 88034 SIVmac/32H + + 29.6 1,490 21.6 960

28.81,57030.0++SIVmac/32H 1,6103738 SIVmac/32H + + 24.7 1,980 11.0 36039 SIVmac/32H + 52.0+ 37.3 2,960 3,930

1 Strain of virus inoculated at the time of infection. All monkeys received intravenously 40 MID50 of SIVmac251/32H.2 Anti-SIV antibodies in plasma as detected by ELISA immunoassay as described previously [38].3 Virus isolation was performed by co-culturing monkey PBMCs (3×106) with human CEMX174 human cell line (1×106).The co-cultures were monitored for the presence of SIV p27gag antigen in the supernatants for 30–40 days as describedpreviously [38].4 Thd treatment started at 10 months after infection with SIVmac251/32H.5 Absolute counts of circulating CD4+ cells/mm3.

4

Thalidomide therapy during SIV infection

tion was analyzed after the 1st cycle of Thd, weobserved a positive SIV peptide-stimulated prolif-eration in none of the monkeys in the placebogroup and in three (37.5%) of the Thd-treatedanimals (Fig. 1, panels A,C). None of the monkeysin the placebo group showed a positive prolifera-tion to either Flu or SIV peptides before the 2ndcycle. In contrast, one of the Thd-treated monkeysretained a positive proliferation to both Flu andSIV peptides. Finally, after the 2nd cycle, one(14%) and two (28%) animals in the placebo groupshowed proliferation to Flu and SIV peptides, re-spectively, whereas the PBMCs of two (25%) andof four (50%) animals in the Thd-treated prolifer-ated to Flu and SIV peptides, respectively (Fig. 1,panels B,D).

Mitogen-stimulated TNF-a production

Mitogen-stimulated TNF-a production by PBMCswas measured in both groups of monkeys beforeand after the two cycles of therapy (Fig. 2). TNF-aproduction (pg/ml) before the 1st cycle was151.9981.2 and 347.29133.1 (mean9SD) in themonkeys subsequently receiving placebo or Thd,respectively. TNF-a production after the 1st cycleof therapy was 99.3941.2 in the placebo groupand 167.4979.2 (mean9SD) in the Thd-treatedanimals. Although the high SD of the levels ofTNF-a between the two groups of monkeys makesstatistical comparison of these values difficult, theinhibition of TNF-a production appears, however,more evident at the end of the 2nd cycle of treat-ment with Thd [96.3922.2 in the placebo groupvs. 23.5919.7 (mean9SD) in the Thd-treatedanimals].

Baseline expression of CD28 receptor on CD4+ andCD8+ T-cell subsets in SIV-infected and naive macaques

Using a three-color fluorescence, PBMCs from 15SIV-infected and eight naive macaques were ana-lyzed for CD4+CD28+ and CD8+CD28+ sub-sets. The percentage of cells positive for CD28receptor among the SIV-infected macaques variedfor each individual macaque from a minimum of12% to a maximum of 76% on CD4+ lymphocytes(range of CD4+ cell/mm3 260–2,640) and from aminimum of 17% to a maximum of 99% on CD8+

lymphocytes (range of CD8+ cell/mm3 930–4,370), with a mean percentage of 44 and 47%,respectively (Fig. 3). The percentage of expressionof CD28 on CD4+ and CD8+ T-cells of naivemonkeys was 86 and 36%, respectively. Compari-son between values from SIV-infected and naivemonkeys revealed that infected monkeys are char-

5

Tabl

e2.

Com

paris

onof

the

effe

cts

ofTh

dtre

atm

ent

onvir

olog

ical

para

met

ers

oftre

ated

vs.

plac

ebo

SIV-

infe

cted

cyno

mol

gus

mac

aque

s

Thd-

trea

ted

Pla

cebo

-tre

ated

Day

0D

ay7

Day

71D

ay14

2D

ay0

Day

14

Mon

key

code

RN

AV

LR

NA

VL

RN

AM

onke

yco

deV

LR

NA

VL

VL

RN

AV

L4R

NA

3

0.01

22.5

0.01

B1

ND

500.

01B

10.

01B

1N

D45

3944

0.30

355

0.28

11N

D2.

843

0.01

900.

0145

ND

22.5

380.

1822

.50.

15B

1N

D45

150.

3035

50.

2611

ND

5.7

1411

ND

B1

0.65

900.

4333

45N

D11

0.30

450.

3227

0.50

900.

75B

1N

D90

0.25

178

0.29

3611

ND

178

3716

0.71

355

0.78

11N

D90

340.

9835

50.

955.

7N

D22

.520

355

ND

450.

6890

0.70

22.5

ND

5.7

180.

4317

80.

4811

0.62

5.7

0.65

2390

ND

1E

ndof

the

1st

cycl

eof

ther

apy.

2E

ndof

the

2nd

cycl

eof

ther

apy.

3P

lasm

aS

IV-R

NA

asde

term

ined

byqu

alita

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say:

The

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pres

sed

asA

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dete

rmin

edby

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etric

anal

ysis

(rang

e0.

01–4

AU

)oft

heba

ndco

rres

pond

ing

atth

eam

plifi

edre

gion

ofth

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IVga

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4C

ell-a

ssoc

iate

dvi

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Num

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06P

BM

Cs.

ND

=no

tdo

ne.

Di Fabio et al.

acterized by a sharp reduction of the CD28 recep-tor on CD4+ T-cell subsets (44 vs. 86% SIV-in-fected vs. normal, respectively) and a slightincrease on CD8+ T-cell subsets (47 vs. 36% SIV-infected vs. normal, respectively) (Fig. 3).

Up-regulation of CD28 receptor on CD4+ T-cells

The baseline mean percentage value (before treat-ment) of CD4+CD28+ T-cells was 36% in thegroup that received Thd and 53% in the monkeysreceiving placebo. The results are expressed (meanvalue) as a global analysis of Thd therapy insteadof presenting an analysis of individual monkeys.After the 1st cycle of treatment (7 days), Thd-treated macaques showed a significant increase inthe percentage of CD4+CD28+ subset from 36 to70% (PB0.05) (Fig. 4, panel A) while the placebogroup did not show significant difference (from 53to 61%; P\0.05) (Fig. 4, panel B). The meanpercentage value of CD4+CD28+ T-cells at thestart of the therapy was different between theThd-treated (36%) and the placebo group (53%)depending on the inter-individual variability foundamong the SIV-infected animals. When the expres-sion of the CD28 receptor on CD4+ T-cells wasmeasured before and after the 2nd cycle of Thdadministration (t01 vs. t14), no relevant modificationwas detected (P=0.7764). However, when theanalysis was performed comparing the percentageof CD4+CD28+ T-cells between treated vs.placebo on day 0 (t0) and on day 14 (t14), a signifi-cant increase of CD28 expression on CD4+ T-cells(from 36 to 71%) was found for the treated group(PB0.05), while the increase from 53 to 62% thatoccurred in the placebo group was not significant.

These results are important because the in-creased expression of the CD28 receptor in Thd-

treated monkeys was concomitant to the decreaseof absolute CD4+ T-cells number from 1,490(range 850–2,080) to 844 cells/mm3 (range 290–1,510). A similar decrease from 1520 (range 260–3,930) to 871 cells/mm3 (range 310–2,410) (Fig. 4)also occurred in the control monkeys. The decreaseof the CD4+ T-cell number during the course ofthe experiment (P=0.0015, t0 vs. t14) in bothgroups is important in that it was not influenced bythe Thd treatment but rather it seems to follow theprogression of infection to clinical symptoms ofthe disease. In fact, the number of CD4+ circulat-ing T-cells measured 7 weeks after the 2nd cycle oftherapy was 611 cells/mm3 (range 10–1,150) in theThd-treated monkeys and 918 cells/mm3 (range10–3,150) in the placebo group (data not shown).

Down-regulation of CD28 on CD8+ T-cells

The baseline percentages CD8+CD28+ T-cellswere 57 and 35% comparing the Thd vs. placebogroup, respectively. Among the seven controls re-ceiving placebo, the percentage of CD8+ T-cellsthat expressed the CD28 receptor was 35% andincreased up to 56% during the first phase of theexperiment (PB0.05). On the contrary, the per-centage of CD8+CD28+ T-cells decreased from 57to 23% (PB0.05) after 7 days of Thd treatment ofthe eight SIV-infected macaques (Fig. 4, panel C).The 2nd cycle of treatment (t01 vs. t14) did not showsignificant modification of CD28 on CD8+ T-cells.Nevertheless, the monkeys treated with Thd, ac-cording to the analysis of CD8+CD28+ T-cellsperformed on day 0 (t0, 1st cycle) and on day 14(t14, end of 2nd cycle), revealed a significant de-crease of CD28 receptor from 57 to 22% (PB0.05). On the contrary, no modification (from 35 to33%) was observed among placebo-treated mon-

Fig. 1. Proliferative responses to SIV peptides.Each bar represents a single animal. Four differ-ent time points are shown: day 0 and day 7 (1stcycle), day 0 and day 14 (2nd cycle). The prolifer-ative responses of PBMCs from Thd-treated mon-keys are indicated in panels A (1st cycle) and B(2nd cycle). The proliferative responses of PBMCsof placebo-treated monkeys are reported in panelsC (1st cycle) and D (2nd cycle). The dashed lineindicates the threshold limit (mean plus 5-fold theSD of non-stimulated PBMCs) used to discrimi-nate positive from negative samples.

6

Thalidomide therapy during SIV infection

Fig. 2. Mitogen-stimulated TNF-a production from PBMCs of Thd-treated monkeys (panel A) and of placebo-treated controls(panel B). Each line represents a single animal. Four different time points are shown: pre-treatment×2 and post-treatment×2.Thd-treated monkeys underwent two sequential cycles of therapy; placebo controls were bled at the same time points.

keys (Fig. 4, panel D). At the same time, a signifi-cant expansion of the CD8+/CD28− T-cell popu-lation (data not shown) was observed in monkeysthat received Thd. The absolute CD8+ T-cellsnumber was counted at the beginning and at theend of the experiment in both groups and a reduc-tion of CD8+ T-cell subsets was observed in theThd-treated macaques from 2,880 (range 1,500–4,370) to 2,110 cell/mm3 (range 1,800–3,010) andin the placebo group from 2,390 (range 1,590–3,280) to 1,800 cell/mm3 (range 660–2,660). There-fore, the Thd treatment did not affect the absolutenumber of CD8+ T-cells because the decrease ofabsolute CD8+ T-cells (P=0.0022, t0 vs. t14) oc-curred in both groups (Fig. 4).

Discussion

The immunomodulatory effects of Thd have re-ceived renewed attention since they were first de-scribed in the 1960s [9]. A number of studies wereinitiated to develop analogues of Thd with in-creased anti-TNF-a inhibitory activity but withoutthe teratogenic effects. It has become clear thatThd and its analogues present a wide and in somecases opposite immunomodulatory effect [8, 18].

In this study, we report that Thd treatment isassociated with a decreased capability of monkeyPBMCs to produce TNF-a after in 6itro mitogenstimulation and with a restoration of the prolifera-tive response to specific SIV peptides in 6itro. Pre-vious reports indicated that chronic exposure toTNF-a impairs the activation through the T-cellreceptor (TCR)/CD3 complex and that treatmentwith anti-TNF-a neutralizing antibodies restoresthe proliferative responses of PBMCs to mitogensand recall antigens [7]. Furthermore, Thd in addi-

tion to its known TNF-a inhibitory activity is ableper se to stimulate in 6itro the primary humanT-cell responses that are dependent on the au-tocrine production of interleukin-2 (IL-2) and in-terferon (IFN)-g in the presence of aTCR-mediated signal [8, 17]. Thus, to achieve opti-mal activation, in addition to the primary T-cellsignal mediated by the T-cell Ag receptor, a co-stimulus is necessary. We report here that therestoration of T-cell function is associated with theincreased expression of the CD28 cell surfacemarkers on CD4+ cells of SIV-infected monkeysafter Thd treatment. The CD28 is the co-receptor

Fig. 3. Baseline expression of CD28 receptor in PBMCs ofeight naive and 15 SIV-infected monkeys. Triple-color immu-nofluorescence was used to determine the percentage ofCD28+ subsets within the CD4+ and CD8+ subsets. Eachbar represents the total number of CD4+ or CD8+

lymphocytes. The white areas represent the number of CD28+

cells within each group together with the corresponding per-centage (%) subset of total CD4+CD28− (a) orCD8+CD28− ( ) lymphocytes.

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Di Fabio et al.

Fig. 4. Up- or down-regulation of CD28 receptor within the total CD4+ (panels A and B) or total CD8+ (panels C and D) T-cellsof Thd-treated (panels A and C) and placebo-treated monkeys (panels B and D). Placebo- and Thd-treated monkeys were bled atthe same time points on day 0 (before treatment), on day 7 (after the 1st cycle of treatment) and on day 14 (after the 2nd cycle oftreatment). Each bar represents the absolute CD4+ cell number composed by CD4+CD28− T-cells (b) and by CD4+CD28+

T-cells ( ) (panels A and B) or the absolute CD8+ cell number composed by CD8+CD28− T-cells (a) and by CD8+CD28+

T-cells ( ) (panels C and D). In the white portion of each bar is reported the percentage (%) of CD4+CD28+ or CD8+CD28+

T-cells. P values refer to multiple comparison of t0 values vs. values after treatment within Thd-treated (left panels) andplacebo-treated (right panels) animals.

for the second signal from professional antigenpresenting cells which use CD80 or CD86 (B7-1 orB7-2) for the second signal [1, 19, 37]. A number ofreports emphasize the role of the CD28 molecule inregulating T-cell proliferation, in maintaining T-cell responsiveness in 6itro with the expression ofIL-2 [26], and in triggering human natural killer(NK) cell-mediated cytotoxic effects on B-7 posi-tive tumor cells [14]. A reduced CD28 cell surfacereceptor expression has been observed in thePBMCs of patients infected with HIV showing areduced responsiveness to mitogens and it wasfound to correlate with virus replication andspread [16]. The stimulation in 6i6o of CD28 mightbe useful when immune stimulation is a desirableoutcome of a therapeutic approach, as in the caseof patients having viral infections or tumors. Onthe other hand, because CD28 contributes to thepathogenesis of the graft vs. host disease, interven-tion with agents that block CD28 function mighthave a beneficial effect [41]. Thus, the CD28 recep-tor has been identified as an important target fortherapeutic approaches [31]. Importantly, the up-regulation of the CD28 receptor on CD4+ monkeycells is paralleled by a simultaneous depletion of

the absolute number of CD4+ cells that is non-de-pendent on Thd treatment. In fact, the number ofCD4+ cells observed at the end of the 1st cycle,when measured at an additional time point post-therapy, did not return to the pre-treatment level.We previously examined the effect of Thd on acti-vation markers and cytokine production in earlyHIV infection and we did not note significantchanges in the immune activation status. The mostdramatic activity of Thd has been observed on oraland gut mucosa in the latest stages of HIV diseasewhere the inhibition of TNF-a decreases cachexiaand leads to moderate weight gain [27]. In 6itroinvestigations have shown that Thd can modifysurface integrin receptors on leucocytes [30] anddown-regulate the CD26 receptor in naive volun-teers and macaques [29].

In the present study, we demonstrated that Thd,although able to inhibit TNF-a production, up-regulate the CD28 receptor on CD4+ cells andrestore the proliferative responses, failed, however,to activate the CD8+ T-cells and to increase theexpression of CD28 receptor on CD8+ T-cells.Our data are in line with those suggesting that Thdmight have a differential effect on CD4+ and

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Thalidomide therapy during SIV infection

CD8+ cells depending on the presence of CD4+

cells [17] and that through the inhibition of TNF-aproduction it might modulate the T-cell receptorssuch as CD28 [6], which in turn is able to preventapoptosis through the up-regulation of Bcl-XL [25].It is well known that CD8+/CD28+ cells are re-sponsible for the non-cytotoxic antiviral activityconsisting of inhibition of HIV replication [24]whereas cells showing the CD8+/CD28− pheno-type have been reported to play an important rolein antiviral cytotoxic lymphocyte (CTL)-mediatedactivity [13]. Having observed an expansion of theCD8+/CD28− cells in monkeys treated with Thd,one could expect an inhibition of the viral loadmediated by CTL or NK cells. The lack of antiviralactivity might be explained by the fact that CD8+

cells become non-responsive after activation in thepresence of co-stimulation [10]. Other results specu-lated that CD4+ activated T-cells not only helpCD8+ T lymphocytes to mature into effector Killercells but can also limit their growth and functionsvia Fas/Fas ligand-mediated apoptosis [33]. In linewith these considerations, but in contrast to thesmall increase of the viral load observed in Thd-treated human patients [22], significant variationson the viral load were not detected in our samples.This suggests that a reduction in the activation ofCD8+ cells might be a desirable outcome in HIVinfection.

These data seem to suggest that Thd exhibits acascade of complex immunomodulatory functionsother than the inhibitory effects on TNF-a produc-tion. An understanding of these functions gainedthrough the use of animal models is critical forfuture applications of either Thd or its analogues asadjuvant on therapeutic approaches in human pa-tients infected with HIV.

AcknowledgmentsWe thank Dr. Cranage and Dr. Greenaway, PHLS Centrefor Applied Microbiology and Research, Porton Down, UK,for providing the virus (SIVmac251/32H) through the Pro-gram EVA of the EC Program on AIDS research, directedby H. Holmes. We also thank Miss A. Lippa, Mrs. F.M.Regini and Miss A. Neri for editorial assistance and F.Varano, F. Incitti, P. Di Zeo, S. Fazzitta, M. Chiodi, A.Marini, D. Silvani, S. Alessandroni and R. Marinelli forhandling the cynomolgus colony.

This work was supported by a grant from the ‘AnimalModel Development Project’ of the Italian Ministry ofHealth, Rome, Italy.

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