Proc. Natl. Acad. Sci. USAVol. 93, pp. 3126-3131, April 1996Immunology

Inhibition of acute in vivo human immunodeficiency virusinfection by human interleukin 10 treatment of SCIDmice implanted with human fetal thymus and liverTOBIAS R. KOLLMANN*, MASSIMO PETTOELLO-MANTOVANIt, NIKOS F. KATOPODIS*, MOSHE HACHAMOVITCHt,ARYE RUBINSTEIN*t, ANA KIMt, AND HARRIS GOLDSTEIN*t§Departments of *Microbiology and Immunology, tPediatrics, and *Gynecology and Obstetrics, Albert Einstein College of Medicine, Bronx, NY 10461

Communicated by Stanley G. Nathenson, Albert Einstein College of Medicine, Bronx, NY, December 29, 1995 (received for reviewNovember 2. 1995)

ABSTRACT To improve the usefulness of in vivo modelsfor the investigation of the pathophysiology of human immu-nodeficiency virus (HIV) infection, we modified the construc-tion of SCID mice implanted with human fetal thymus andliver (thy/liv-SCID-hu mice) so that the peripheral blood ofthe mice contained significant numbers of human monocytesand T cells. After inoculation with HIV-159, a primary patientisolate capable of infecting monocytes and T cells, the modi-fied thy/liv-SCID-hu mice developed disseminated HIV in-fection that was associated with plasma viremia. The devel-opment of plasma viremia and HIV infection in thy/liv-SCID-hu mice inoculated with HIV-I59 was inhibited by acutetreatment with human interleukin (IL) 10 but not with humanIL-12. The human peripheral blood mononuclear cells in thesemodified thy/liv-SCID-hu mice were responsive in vivo totreatment with exogenous cytokines. Human interferon yexpression in the circulating human peripheral blood mono-nuclear cells was induced by treatment with IL-12 and inhib-ited by treatment with IL-10. Thus, these modified thy/liv-SCID-hu mice should prove to be a valuable in vivo model forexamining the role of immunomodulatory therapy in modify-ing HIV infection. Furthermore, our demonstration of the invivo inhibitory effect of IL-10 on acute HIV infection suggeststhat further studies may be warranted to evaluate whetherthere is a role for IL-10 therapy in preventing HIV infectionin individuals soon after exposure to HIV such as for childrenborn to HIV-infected mothers.

Investigation of the kinetics of the development of resistanceby human immunodeficiency virus (HIV) to antiviral agents inHIV-infected individuals has indicated that the clinical courseof HIV-mediated disease involves a dynamic interaction be-tween rapidly replicating virus and the immune system (1, 2).The plasma of HIV-infected individuals contains the mostrecently replicated HIV, therefore, the population dynamics ofHIV replication are best evaluated by examining the levels ofHIV present in the plasma (3). Thus, the development of anin vivo animal model wherein plasma HIV levels can bemonitored would greatly facilitate the ability to evaluate the invivo efficacy of therapeutic interventions. An attractive smallanimal model for studying in vivo HIV infection is SCID-humice, SCID mice implanted with human fetal thymus and liver(4). The usefulness of this model has been limited by the lownumbers of circulating human T cells and the absence ofdetectable levels of human monocytes in the peripheral bloodof these SCID-hu mice (5). We recently described modifica-tions in the construction of SCID-hu mice that significantlyincreased the numbers of human T cells present in theirperipheral blood, spleen, and lymph nodes and permitted the

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

development of disseminated HIV infection after peripheralinoculation with multiple strains of HIV-1 including monocy-totropic strains (6, 7). In the present study, we report that theperipheral blood of these modified SCID-hu mice is alsopopulated with significant numbers of human monocytes thatcan potentially be infected with HIV. Furthermore, we dem-onstrate that after HIV infection, these mice develop plasmaviremia that was markedly inhibited by treatment with inter-leukin (IL) 10.

MATERIALS AND METHODS

Cytokines and Antibodies. Human recombinant IL-12 was agift from Stanley Wolf (Genetics Institute, Cambridge, MA)and human recombinant IL-10 was provided by SatwantNarula (Schering-Plough Research Institute, Kenilworth, NJ).Peridinin chlorophyll protein-conjugated monoclonal antibod-ies to human CD45, fluorescein isothiocyanate-conjugatedmonoclonal antibodies to human CD4 and CD14, and phyco-erythrin-conjugated monoclonal antibodies to human CD8and CDllb were obtained from Becton Dickinson.

Implantation of Human Fetal Thymic and Liver (hu-thy/liv) Tissue into SCID Mice. The hu-thy/liv tissue wasobtained from fetuses at 17-21 gestational weeks within 8 hafter the elective termination of pregnancy and implanted intoSCID mice (6-8 weeks old) as described (6, 7). Briefly, thehu-thy/liv tissue obtained from one fetus was kept on ice andcut into 1-mm3 pieces. After the SCID mice were anesthetizedwith pentobarbital (40-80 mg/kg), the left and right kidneyswere sequentially exteriorized and subcapsularly implantedwith at least 10 pieces of hu-thy/liv tissue (resulting in thy/livSCID-hu mice). This procedure resulted in minimal postop-erative morbidity and mortality and the successful implanta-tion of >95% of the mice. The implanted tissue increased by>20-fold from its original size by 3 months after implantation.The consent forms and procedures used in this study werereviewed and approved by the Albert Einstein College ofMedicine Committee on Clinical Investigation.Flow Cytometric Analysis. Mononuclear cells were har-

vested from the peripheral blood of the thy/liv-SCID-hu miceand stained with phycoerythrin-, fluorescein isothiocyanate-,or peridinin chlorophyll protein-conjugated mouse monoclo-nal antibodies that were specific for human (and negative formouse) CD45, CD4, CD8, CD14, or CDllb as described (7, 8).

Abbreviations: HIV, human immunodeficiency virus; thy/liv-SCID-hu mice, SCID mice implanted with human fetal thymus andliver; hu-thy/liv, human fetal thymus and liver; RT-PCR, reversetranscription-coupled polymerase chain reaction; X32-m, 132-microglobulin; IL, interleukin; IFN-y, interferon-y; PBMC, peripheralblood mononuclear cell; TCID, tissue culture infective dose; TNF-a,tumor necrosis factor a.

§To whom reprint requests should be addressed at: Albert EinsteinCollege of Medicine, Chanin Building, Room 601, 1300 Morris ParkAvenue, Bronx, NY 10461.

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The peripheral blood of the SCID mice used contained about2.0 x 105 cells per ml and the percentage of human CD4 andCD8 cells given is the percentage of cells present in thelymphocyte gate while the percentage of human CD14 andCDlb cells indicated is the percentage of cells in the mono-cyte gate. Expression of the human surface proteins by leu-kocytes present in the SCID-hu mice was assessed by three-color flow cytometric analysis using a FACScan cell analyzerwith LYSIS-II software (Becton Dickinson) with expression ofhuman CD45 used to confirm the human origin of the cells.Lymphocyte and monocyte gates were set on the basis offorward and side scatter profiles to correspond to gates set forcontrol human (from healthy adult volunteer) lymphocytesand monocytes. Nonviable cells and unlysed red blood cellswere gated out based on their forward and side scatter profiles.Single, double, and triple staining of positive and negativecontrol samples (human adult and C.B-17 mouse mononuclearcells) and of the appropriate mouse IgG isotype controls wereassessed and used to compensate for phycoerythrin vs. fluo-rescein isothiocyanate vs. peridinin chlorophyll protein emis-sion and to set cut off values for the quadrants.

Detection of Human Cytokine Gene Expression by ReverseTranscription-Coupled Polymerase Chain Reaction Amplifi-cation (RT-PCR). The pattern of in vivo human cytokineexpression by the human cells present in thy/liv-SCID-hu micewas assessed by using a modification of a previously describedtechnique (6, 8). Briefly, peripheral blood mononuclear cells(PBMCs) from the thy/liv-SCID-hu mice were lysed in gua-nidine isothiocyanate (4 M) buffer, and cellular DNA andRNA were separated by cesium chloride (5.7 M) densitygradient centrifugation and precipitated with ethanol. RNA (7utg) in 7 Aul of double-distilled H20 was mixed with 4 ,l of 5 xbuffer (250 mM Tris-HCl, pH 8.3/375 mM KCl/15 mMMgCl2), 2 ,tl of dithiothreitol (100 mM), 1 ,tl of randomhexamers (BRL-GIBCO), and 5 ,l of mixed dNTPs (each at2 mM), heated to 65°C for 10 min, and cooled on ice for 5 min,and then 1 ,l (200 units) of Superscript reverse transcriptase(BRL-Gibco) was added. This final reaction mixture wasmixed, briefly centrifuged, incubated at 37°C for 60 min, andthen placed on ice. After reverse transcription of total RNA (7jig) extracted from the PBMCs of the thy/liv-SCID-hu mice,cDNA was amplified by PCR with human-cytokine-specificprimers for 60 cycles of denaturation at 94°C for 1 min,annealing at 65°C for 1 min, and extension at 72°C for 1 min.The presence of the target mRNA was indicated by thevisualization of an amplification product of the predicted sizeafter fractionation of the PCR products by electrophoresis andethidium bromide staining. The nucleotide sequences for the5' and 3' primer pairs for each cytokine were selected frompublished DNA sequences and were designed to be human-specific and mRNA-specific as described (6, 8). All sampleswere analyzed by RT-PCR for the presence of human 32-microglobulin (f32-m) to verify the integrity of the samplemRNA and the efficiency of subsequent reverse transcription,and positive and negative controls were always included.

Detection of HIV RNA in the Mouse Plasma. The thy/liv-SCID-hu mice were infected either by direct injection of 300tissue culture 50% infective dose (TCID50) units of HIV-159 in30 Al into the left hu-thy/liv implant or by i.p. injection of 8000TCID50 units of HIV-159 in 800 ,ul as described (7). The plasmawas isolated from the blood of the thy/liv-SCID-hu mice andanalyzed for the presence of HIV RNA by RT-PCR using amodification of a previously described technique (6). RNA wasextracted from plasma (100 ,l) by solubilization in guanidineisothiocyanate (4 M) buffer and precipitation with isopropra-nol followed by treatment with RNase-free DNase (BRL-GIBCO). The RNA was reverse-transcribed as describedabove, the cDNA was amplified for 35 cycles with a primer pairspecific for the gag gene segment (S38/39), electrophoresedthrough 1.5% NuSieve/0.5% SeaKem agarose (FMC) gel, and

subjected to Southern blot analysis with a digoxigenin-labeledinternal probe, SK19, specific for the SK38/39 product. Thehybridized probe was detected by using the Genius luminescentdetection kit (Boehringer Mannheim).A given sample was scoredas positive ifPCR amplification resulted in a DNA product of thepredicted size that hybridized to the specific internal probe.Positive and negative controls were always included.

Titration of HIV-Infected Mononuclear Cells in the hu-thy/liv Implant by Limiting Dilution Coculture. The titer ofHIV-1-infected mononuclear cells present in the hu-thy/livimplants was determined as described (7). Mononuclear cellsisolated from the hu-thy/liv implant in 1:5 dilutions rangingfrom 1 x 106 cells to 3.2 x 101 cells were cultured inquadruplicate for 1-2 weeks at 37°C in 24-well culture plateswith 1.0 x 106 phytohemagglutinin-activated donor mononu-clear cells in a total volume of 2.0 ml of RPMI 1640 mediumcontaining 10% (vol/vol) fetal calf serum and IL-2 (32 units/ml). The p24 antigen content of the culture supernatant wasthen measured by using the HIV-1 p24 core profile ELISAassay (DuPont/NEN). The lowest number of added mononu-clear cells that infected at least half of the quadruplicatecultures with HIV-1 was taken as the end point or TCID andthe data are presented as the number of TCID units per 106mononuclear cells.

Statistical Analysis of the Data. The 2 x 2 Fisher exact testand the Fisher's exact test were used to assess the statisticalsignificance of the data.

RESULTSThe Peripheral Blood of These Modified thy/liv-SCID-hu

Mice Is Populated with Significant Numbers of Human TCells and Monocytes. We have shown (6, 7) that by increasingthe quantity of tissue implanted in the SCID mice to at leastten 1-mm3 pieces of hu-thy/liv tissue and by implanting thisamount of tissue under both kidney capsules, we could mark-edly enhance the peripheral engraftment of thy/liv-SCID-humice with human T cells. The peripheral blood of the thy/liv-SCID-hu mice (n = 26) constructed in this fashion waspopulated 3 months later with 13 ± 2.5% human CD4+ T cellsand 3.2 ± 0.4% human CD8+ T cells (mean ± SEM). Inaddition to providing a source of pre-T cells capable ofmigrating into and maturing in the adjacent human thymictissue, the implanted human fetal liver also contains stem/precursor cells that are capable of maturing into the myeloidlineage (9). Therefore, to determine whether myeloid cellsdifferentiating in the implanted fetal liver were capable ofpopulating the peripheral blood of our modified thy/liv-SCID-hu mice with human monocytes, PBMCs of these micewere evaluated for the expression of human CD45 and themonocyte-associated markers CD14 and CD11llb. A distinctpopulation of human CD45+ CD14+ and CD45+ CDllb+monocytes was present in the peripheral blood of the thy/liv-SCID-hu mice (Fig. 1). In eight thy/liv-SCID-hu mice exam-ined, the peripheral blood monocyte gate contained 2.6 ±1.7% and 3.1 ± 1.9% human CD45+ CD14+ and CD14+CDllb+ cells, respectively. Thus, the peripheral blood of ourmodified thy/liv-SCID-hu mice was populated with significantnumbers of human T cells and monocytes.

IL-10 Treatment Inhibits in Vivo HIV Infection. The resultsof in vitro studies have indicated a dichotomy between theeffects of different cytokines on HIV replication, with IL-10reported to inhibit HIV replication in monocytes (10, 11) andIL-12 shown to enhance HIV infection in PBMCs (12).Therefore, we used the thy/liv-SCID-hu mice to investigate theeffect of IL-10 or IL-12 treatment on the in vivo course ofinfection after inoculation with a primary patient isolate ofHIV, HIV-159, previously characterized as being able to infectprimary monocytes and lymphocytes and being nonsyncytiuminducing (7). To determine whether cytokine treatment could

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of thy/liv-SCID-hu mice at least 3 months after implantation, cells in the monocyte gate were analyzed by three-color flow cytometry for theexpression of human CD45, CD14, and CDllb. A representative dot histogram is shown with the percentages of cells in each quadrant indicated.

inhibit subsequent HIV infection, thy/liv-SCID-hu mice weretreated on alternate days with IL-10 (10 jig per dose), IL-12 (10gug per dose), or phosphate-buffered saline (PBS), with twodoses before infection and then with seven doses after HIV-159infection. Two days after the last dose, the degree of activeHIV replication was assessed by using RT-PCR to examine themice for the presence of plasma viremia (Fig. 2 A and B).Whereas plasma viremia was detected in 7 of 10 thy/liv-SCID-hu mice treated with PBS, and 2 of 2 thy/liv-SCID-humice treated with IL-12, plasma viremia was detected in noneof the 6 thy/liv-SCID-hu mice treated with IL-10 (P = 0.011).IL-10 treatment also markedly decreased the extent of HIVinfection detected in the hu-thy/liv implants of the thy/liv-SCID-hu mice 2 days after the last dose. In the PBS-treated i.p.inoculated thy/liv-SCID-hu mice, all of the hu-thy/liv implants

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were significantly infected with HIV with a mean of 2454TCID units per 106 mononuclear cells (n = 4) compared to theIL-10-treated i.p. injected thy/liv-SCID-hu mice, where noHIV infection was detected in hu-thy/liv implants from threemice and only minimal HIV infection (5 TCID units per 106mononuclear cells) was detected in the hu-thy/liv implants ofthe fourth mouse. Similar results were observed for intra-implant inoculated thy/liv-SCID-hu mice, where markedly lessviral load was present in the thy/liv implants of IL-10-treatedmice (mean = 475 TCID units per 106 mononuclear cells; n =

2) compared to the PBS-treated mice (mean = 28,601 TCIDunits per 106 mononuclear cells; n = 6).

IL-10 also inhibited HIV infection when treatment wasstarted a short time after inoculation. The thy/liv-SCID-humice were infected with HIV by intraimplant inoculation and

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FIG. 2. Detection of HIV RNA in the plasma of PBS-, IL-10-, and IL-12-treated thy/liv-SCID-hu mice after inoculation with HIV. (A) Thethy/liv-SCID-hu mice were pretreated on alternate days for two doses with PBS (n = 4) or IL-10 (n = 4), injected i.p. with HIV-159 (8000 TCID50units), and then treated on alternate days for seven doses with PBS or IL-10. (B) After thy/liv-SCID-hu mice were pretreated on alternate daysfor two doses with PBS (n = 6), IL-12 (n = 2), or IL-10 (n = 2), HIV-159 (400 TCID50 units) was injected into the hu-thy/liv implant, and thenthe mice were treated on alternate days for seven doses with PBS, IL-12, or IL-10. (C) Eleven days after HIV-159 (300 TCID50 units) was injectedinto the hu-thy/liv implants of the thy/liv-SCID-hu mice, the mice were treated with PBS (n = 3), IL-10 (n = 2), or IL-12 (n = 2) on alternatedays for seven doses. (D) Six weeks after HIV-159 (8000 TCID50 units) was injected i.p. into thy/liv-SCID-hu mice, the mice were treated with PBS(n = 3), IL-10 (n = 3), or IL-12 (n = 2) on alternate days for seven doses. Two days after the last dose of cytokine, the presence of HIV in theplasma of the thy/liv-SCID-hu mice was then assessed.

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11 days later the mice were started on treatment with alternateday injections of IL-10 (10 gjg per dose), IL-12 (10 jg perdose), or PBS for a total of seven doses. HIV was detected inthe plasma of two of three thy/liv-SCID-hu mice treated withPBS, two of two thy/liv-SCID-hu mice treated with IL-12, andin none of the two thy/liv-SCID-hu mice treated with IL-10(Fig. 2C). Furthermore, while the hu-thy/liv implants of thePBS-treated thy/liv-SCID-hu mice contained a mean of 4191TCID units per 106 mononuclear cells, the hu-thy/liv implantsof the IL-10-treated thy/liv-SCID-hu mice contained a meanof 375 TCID units per 106 mononuclear cells. However, whentreatment of the thy/liv-SCID-hu mice was started 6 weeksafter infection with HIV-159, HIV viremia was detected in allof the PBS (n = 3), IL-12 (n = 2), and IL-10 (n = 3)-treatedthy/liv-SCID-hu mice (Fig. 2D). Significant infection in thethy/liv implant of the IL-10-treated mice was seen (mean =

14,559 TCID units per 106 mononuclear cells) that was com-parable to that observed in the PBS-treated mice (mean =

28,944 TCID units per 106 mononuclear cells). This indicatedthat a short course of IL-10 treatment at the doses used waseffective in inhibiting HIV infection only when given before orsoon after inoculation.

Effect of IL-10 and IL-12 Treatment of hu-thy/liv-SCID-huMice on Human Interferon y (IFN-y) Gene Expression. Thepresence of significant numbers of human mononuclear cellsin the peripheral blood of our thy/liv-SCID-hu mice combinedwith the development of primer pairs specific for humancytokine gene sequences permitted us to assess the in vivoexpression of human cytokine genes by the human PBMCscirculating in these thy/liv-SCID-hu mice (6, 8). After isolationof PBMCs from the peripheral blood of five thy/liv-SCID-humice, RNA was extracted and evaluated with human-specificRT-PCR for the expression of mRNA encoding TH1-associated cytokines IFN-y, IL-2 and tumor necrosis factor a

(TNF-a) and TH2-associated cytokines IL-4, IL-5, IL-6, andIL-10 (13). While mRNA for human TNF-a, IFN-y, IL-5, andIL-2 was detected in five of five, three of five, three of five, andone of five thy/liv-SCID-hu mice, respectively, mRNA expres-sion for IL-4, IL-6, and IL-10 was not detected. No correlationbetween expression of human cytokine genes and the numberof human PBMCs in the peripheral blood was observed.Detection of human IFN-ymRNA in the PBMCs of only someof the thy/liv-SCID-hu mice allowed us to divide the thy/liv-SCID-hu mice into two groups, one group of mice havingcirculating human PBMCs that expressed human IFN-ymRNA and the other group populated with human PBMCsthat did not express human IFN-y mRNA. We took advantageof the fact that in vitro expression of the human IFN-y gene isregulated by IL-10 and IL-12 (14) to determine, by usingqualitative RT-PCR, whether cytokine gene expression in thehuman PBMCs in our modified thy/liv-SCID-hu mice was

responsive to in vivo regulation. The thy/liv-SCID-hu micewere treated daily for 4 days with i.p. injection of PBS, IL-10(10 ,ug per dose), or IL-12 (10 tig per dose), and the humanIFN-y gene expression of their PBMCs before and aftertreatment was evaluated. Whereas human IFN-y mRNA ex-pression remained negative after PBS injection in the PBMCsfrom seven of eight thy/liv-SCID-hu mice, treatment withhuman IL-12 stimulated human IFN-y mRNA expression inthe PBMCs from five of five thy/liv-SCID-hu mice (P = 0.005).In contrast, the PBMCs from seven of seven thy/liv-SCID-humice that initially expressed human IFN-'y mRNA becamenegative after treatment with IL-10, as opposed to the PBMCsfrom one of eight thy/liv-SCID-hu mice injected with PBS (P= 0.005). A representative gel is shown in Fig. 3 demonstratingthat human IFN-y gene expression by human leukocytespresent in the peripheral blood of four thy/liv-SCID-hu mice(mice 1-4) was not changed by treatment with PBS. Incontrast, treatment with IL-10 suppressed the expression ofhuman IFN-y mRNA by the PBMCs of four thy/liv-SCID-hu

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FIG. 3. IFN-y mRNA production in thy/liv-SCID-hu mice treatedwith IL-10, IL-12, or PBS. The expression of human IFN-y mRNA inthe PBMCs of the indicated thy/liv-SCID-hu mice at baseline and afterfour daily injections with PBS, IL-10 (10 ,tg per dose), or IL-12 (10 jigper dose) was determined by RT-PCR. The amplification products ofthe predicted size were visualized in ethidium bromide-stained gels byUV radiation. The presence of human cells and integrity of theRT-PCR for each sample are indicated by the detection of human132-m by RT-PCR with a primer pair specific for human 132-m cDNA.

mice (mice 5-8) and treatment with IL-12 induced the expres-sion of human IFN-y mRNA by the PBMCs of four thy/liv-SCID-hu mice (mice 9-12). Cytokine treatment did not sig-nificantly affect the numbers of human PBMCs present in theperipheral blood of the thy/liv-SCID-hu mice making it un-

likely that cytokine-induced changes in human IFN-y mRNAwas due to changes in the human PBMC population. Thusthese data indicate that human IFN-7y mRNA expression byPBMCs in our modified thy/liv-SCID-hu mice was responsiveto in vivo regulation by IL-10 and IL-12.

DISCUSSIONModifications that we introduced into the construction ofthy/liv-SCID-hu mice, such as increasing the amount of hu-thy/liv implanted and implanting the hu-thy/liv under bothkidney capsules, markedly increased the numbers of human Tcells present in the peripheral blood, spleens, and lymph nodesof the implanted mice (6, 7). In the current report, flowcytometric data demonstrated that the peripheral blood ofthese modified thy/liv-SCID-hu mice was also populated withsignificant numbers of human monocytes. The presence ofhuman monocytes in these modified thy/liv-SCID-hu mice wassuggested by our observation (7) that these mice could beinfected by i.p. inoculation with monocytotropic strains such as

HIV-1ADA-M, HIV-1JR-FL, and HIV-1sF162. The utility of thesemodified thy/liv-SCID-hu mice for investigating the patho-genesis ofHIV infection is markedly increased by the presenceof circulating human monocytes because these mice can beused to evaluate the in vivo effect of therapeutic interventionson HIV infection of monocytes as well as of T cells. This isimportant in examining the in vivo pathogenesis of HIVinfection because macrophage-tropic HIV variants have beenreported to initiate infection after sexual, parenteral, andvertical transmission and because of the role played by mac-

rophages in HIV dissemination and persistence (15, 16).It is likely that the source of HIV plasma viremia observed

in these modified thy/liv-SCID-hu mice is from the highnumbers of human T cells present in the mouse lymphoidtissue wherein we have detected a high level ofHIV replicationafter inoculation with HIV (6). Because the population ofHIVin the plasma fraction represents the most recently replicated

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pool of virus (1-3), we could use the presence of HIV plasmaviremia to evaluate the effect of cytokine therapy on HIVinfection. In vitro studies have indicated that HIV replicationis inhibited in monocytes by IL-10 (10, 11, 17-19) and isinduced in PBMCs by IL-12 (12). Because the human PBMCsin these modified thy/liv-SCID-hu mice were responsive toexogenous human cytokines, we investigated the effect oftreatment with human IL-10 and IL-12 on HIV infection in ourmodified thy/liv-SCID-hu mice. Although the data indicatedthat IL-10 inhibited HIV plasma viremia when given soon afterinfection, the number of mice examined was not large enoughto draw a definitive conclusion. However, the observation thatno HIV viremia was detected in 6 thy/liv-SCID-hu micepretreated with IL-10 while HIV viremia was present in 7 of10 untreated thy/liv-SCID-hu mice was statistically significant(P = 0.011). Furthermore, decreased HIV viral load wasobserved in the hu-thy/liv implants of the thy/liv-SCID-humice pretreated with IL-10 compared to the untreated thy/liv-SCID-hu mice. Thus, to our knowledge, these data are thefirst demonstration of the in vivo effects of cytokine therapy onHIV infection. Although we demonstrated that IL-10 sup-pressed IFN-y mRNA production by human PBMCs in thethy/liv-SCID-hu mice, the inhibitory effects of IL-10 on HIVreplication may be independent of its modulation of IFN--ygene expression. IL-10 has been reported to inhibit in vitroHIV replication by different mechanisms. IL-10 may directlysuppress HIV production either by inhibiting viral replication(18) or by interfering with HIV protein processing (19).Alternatively, IL-10 may indirectly suppress in vitro HIVreplication by inhibiting the autocrine production of TNF-aand IL-6, cytokines reported to enhance HIV replication (10,11). This is an intriguing explanation because we have reported(6) that HIV infection stimulated in vivo expression of TNF-aby human PBMCs in our modified thy/liv-SCID-hu model.

Studies delineating the kinetics of HIV replication in HIV-infected individuals have suggested that T cells are the pre-dominant source of plasma HIV (1-3). While in vitro studieshave indicated that IL-10 had a significant inhibitory effect onHIV replication in monocytes, it did not have a significantsuppressive effect on HIV production by T cells (17, 19).Therefore, it is likely that the IL-10-mediated reduction inplasma viremia and viral load in the hu-thy/liv implant is dueto its effects on the acute infection. This was further indicatedby the observation that IL-10 administration inhibited HIVproduction in our modified thy/liv-SCID-hu mice populatedwith significant numbers of human T cells and monocytes onlywhen given early in the course of infection. This may provideinsights into the pathophysiology of early HIV infection byindicating that while the major source of new virus in chron-ically HIV-infected individuals is from the T-cell population,the predominant location of viral replication and infectionsoon after infection is in the monocyte population. It is alsopossible that the initial HIV infection in the hu-thy/liv implantmay occur in human monocytes present in the implant. Thus,these observations suggest that therapy aimed at blocking HIVreplication in monocytes may be crucial to abort HIV infectionsoon after exposure. However, because the in vivo effect ofIL-10 on HIV replication in T cells may differ from thatobserved in vitro with mitogen-stimulated T cells, we cannotrule out the possibility that IL-10 is inhibiting HIV replicationin T cells in the thy/liv-SCID-hu mice. Although significantHIV infection occurred in the thy/liv-SCID-hu mice, noevidence of a human T-cell immune response directed againstHIV was detected (7). Therefore, it is unlikely that IL-10mediates inhibition of HIV infection in the thy/liv-SCID-humice by stimulating an anti-HIV response such as by inducingHIV-specific cytotoxic T cells. However, the absence of humanimmune responsiveness in these thy/liv-SCID-hu mice maylimit their usefulness in determining the effect of treatmentwith IL-10 on the development of an HIV-specific immune

response in HIV-exposed individuals. Although the thy/liv-SCID-hu model described here represents a significant ad-vance over in vitro studies, results obtained using these miceshould be interpreted with caution pending confirmation withhuman studies.

Because accessory cells are involved in T-cell regulationsuch as the IL-10- and IL-12-regulated control of IFN--yproduction by T cells (14, 20), the presence of monocytes andT cells in the peripheral blood of these mice permitted us toinvestigate the in vivo regulation of cytokine gene expressionby human T cells. We demonstrated that human IFN-,ymRNAexpression was induced in circulating human PBMCs by theadministration of IL-12 and was inhibited by treatment withIL-10. To our knowledge, this is the first demonstration of invivo regulation of human IFN-y gene expression by IL-10 andIL-12. The in vivo effects of these cytokines on IFN-oy mRNAexpression by the human PBMCs in the thy/liv-SCID-hu miceare compatible with the in vitro capacity of IL-12 to induceIFN-y production by resting human peripheral blood lympho-cytes (21) and neonatal human T cells (22) and IL-10 to inhibitin vitro transcription of cytokine genes by human PBMCs (23).It is likely that signals provided by human monocytes presentin our thy/liv-SCID-hu mice were facilitating the regulation ofhuman IFN--y mRNA because IL-10 indirectly suppressesIL-12 production by downregulating accessory-cell class IImajor histocompatibility complex expression, interfering withantigen-presenting-cell costimulatory signaling, and inhibitingthe production of other cytokines (24). In addition, althoughIL-12 alone can stimulate IFN-y production by T cells, IL-12induction of IFN-y is markedly enhanced by the costimulatorysignal delivered through the interaction between CD28 on thesurface ofT cells and B7 present on monocytes (20). Althoughthe inhibition of human IFN-y gene expression by IL-10 maynot be related to its ability to inhibit HIV replication, itindicated that human PBMCs in the thy/liv-SCID-hu micewere responsive to the effects of exogenous IL-10.

Therefore, our modified thy/liv-SCID-hu mice that arepopulated in the peripheral blood with significant numbers ofhuman T cells and monocytes and that develop plasma viremiaafter infection with HIV should prove to be a valuable modelfor examining the role of immunomodulatory therapy inmodifying HIV infection. Furthermore, since phase I trials todetermine the toxicity of IL-10 in humans have been reported(25), our data suggest that further studies may be warranted todetermine whether IL-10 treatment, when added to anti-HIVdrug therapy, may provide potential benefits especially whengiven to individuals soon after exposure to HIV such as inneedle-stick exposures or children born to HIV-infected moth-ers. In addition, the ability to sample the plasma population ofHIV in these modified thy/liv-SCID-hu mice should permittheir use in the study of the in vivo development of resistanceto antiretroviral agents as reported in studies of HIV-infectedindividuals (1, 2).

We thank D. Gebhardt for assistance in the flow cytometry, A.Watford for assistance in animal care, T. C. Chang for performing thestatistical analysis, and J. Berman for review of the manuscript. Flowcytometry was performed in the Flow Cytometry Core Facility andoligonucleotides were synthesized in the Oligonucleotide SynthesisCore Facility supported by the Cancer Center Grant 5P30CA13330.This work was supported by the National Institutes of Health (NationalInstitute of Allergy and Infectious Diseases Centers for AIDS Re-search Grants AI-27741, AI-20671, and HE-53754). M.P-M. was

supported in part by a grant (AIDS project) from the IstitutoSuperiore di Sanita; Ministero della Sanita, Rome, Italy.

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