Viral pathogenesis in hu-PBL-SCID miceDonald E. Mosier

The transplantation of human cells into immunodeficientmice has provided new models for human immune function,infection by pathogenic viruses that grow in lymphocytes orother hematopoietic cells, and development of hematopoieticlineages. SCID mice reconstituted with adult peripheral bloodmononuclear cells (hu-PBL-SCID mice) maintain someestablished immune responses, but do not produce a fullspectrum of primary cellular or humoral responses.Nonetheless, hu-PBL-SCID mice are a valuable tool forstudying primary infection with human immunodeficiencyvirus (HIV) and reactivation of latent Epstein-Barr virus(EBV) infection. This review will summarize findings fromsuch studies.

Key words: animal models / EBV / HIV / viralpathogenesis

©1996 Academic Press Ltd

THERE WAS GREAT EXCITEMENT in the fall of 1988 whenthree different laboratories reported the successfultransplantation of adult or fetal human cells toimmunodeficient SCID or beige.nude.xid mice.1-3

Two of the models, ours1 and McCune’s,2 weremotivated by the desire to create a small animal modelto study HIV infection.4-10 The study by Kamel-Reidand Dick3 had as its goal the development of humanhematopoietic precursors in immunodeficient mousebone marrow.11-13 The transfer of mature peripheralblood mononuclear cells to SCID mice gave hope ofestablishing an adoptively transferred humanimmune response that would reproduce the fullspectrum of human immunity, while the transplanta-tion of hematopoietic progenitors and fetal thymusmight allow the development of lymphoid and otherhematopoietic lineages and generate a stable human–mouse xenochimera. Now, almost a decade later, wesee that the early expectations have been partiallyfulfilled. As is detailed in the rest of this issue, a fullyfunctional human immune response has yet to be

documented, but adoptive transfer of human immu-nity has been demonstrated.14-17 Sustained, multi-lineage engraftment of human hematopoietic cellshas been achieved.13,18-20 As if in answer to theoriginal motivation for their creation, xenotransplantmodels have proven to be remarkably useful forstudying HIV-1 infection. Moreover, mice trans-planted with adult PBL develop EBV-related B-celllymphoproliferative disease21-24 that is remarkablysimilar to a subset of AIDS-associated lymphomas.25,26

The primary infection of hu-PBL-SCID mice withvariants of HIV-1 has given insight into pathogenesisand immune suppression of infection. Likewise, thereactivation of EBV and its in-vivo transformation ofhuman B lymphocytes has provided new insights intothe pathogenesis of B-cell lymphomas.

Persistence of human cells in the hu-PBL-SCIDmodel

The intraperitoneal injection of human peripheralblood mononuclear cells into SCID mice leads to theselective survival and expansion of human CD3+ Tcells, with smaller numbers of human B cells, mono-cytes and NK cells surviving.27,28 CD3+ T cells rapidlyshow signs of activation and selective expansion ofCD45RO+ memory T cells in both the CD4+ andCD8+ subsets. We have recently observed that anti-bodies to CD40 ligand29 block T-cell activation andsubstantially reduce engraftment of human cells inSCID mice (M. Eckert, I. Atencio, D. Mosier, unpub-lished observations), suggesting that T-cell activationis essential for the success of the human graft. Thismay occur because of recognition of mouse xenoanti-gens30,31 on human antigen presenting cells (B cellsor monocytes), although it is not clear that xenoanti-gens are the sole source of T-cell stimulation. We andothers have observed fatal graft-versus-host diseasewhen adult PBL are transplanted to neonatal SCIDmice.32,33 Conversely, human cord blood cells aremore easily engrafted in newborn SCID mice than inadults.32 These observations suggest that antigenicpriming has occurred in adult donors leading to a

From the Department of Immunology, IMM7, The ScrippsResearch Institute, 10666 North Torrey Pines Road, La Jolla, CA92037, USA

seminars in IMMUNOLOGY, Vol 8, 1996: pp 255–262

©1996 Academic Press Ltd1044-5323/96/040255 + 08$18.00/0

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This response leads to lethal graft-versus-host diseasein newborn SCID recipients, but it is required toestablish a successful graft in adult SCID mice. Cordblood T cells, which have few xenoreactive T cells, canonly survive in the more permissive environment ofthe newborn SCID recipient. Although one groupfinds all human T cells to be ‘anergic’ in hu-PBL-SCIDmice30,31 at later time points after engraftment, wefind that human T cells recovered from hu-PBL-SCIDmice at 4–6 weeks after reconstitution are stillresponsive to antigen or anti-CD3 stimulation [(ref27) and B. Torbett, D. Mosier, unpublishedobservations].

Human B cells survive in hu-PBL-SCID mice mainlyas differentiated plasma cells localized in local lymphnodes or cell adhesions to the peritoneal cavity.37

Human immunoglobulin production can occur forup to a year after PBL transplantation, and we haveobserved EBV-related B-cell lymphomas occurring aslate at 9 months after engraftment in some experi-ments. Although small numbers of human monocyte/macrophages persist in local lymphoid tissue, theymay be critical for establishment of HIV-1 infection(see later). There is no evidence for engraftment ofcirculating human stem cells following PBLinjection.

Human cells can be recovered not only from theperitoneal site of injection into hu-PBL-SCID mice,but also from lymph nodes that drain the peritonealcavity, the spleen, and bone marrow. Low numbers ofT cells are present in the peripheral blood and otherlymph nodes. We have not detected human cells inthe thymus, but they are often found in perithymiclymph nodes adherent to the thymic capsule. CD4+ Tcells usually exceed CD8+ T cells in lymph nodes,whereas the reverse is true in cells recovered byperitoneal lavage.

The extensive T-cell activation in the hu-PBL-SCIDmodel provides a ready target for HIV-1 infection, andmimics the extensive lymphocyte activation seen inchronic HIV-1 infection.38,39 Injection of HIV-1 into ahu-PBL-SCID mouse is thus not similar to a needlestick injury in a normal individual, but it is a relevantand important model for HIV-1 research as long asthe underlying biology is understood.

Consequences of HIV-1 infection in thehu-PBL-SCID model

HIV-1 infection of hu-PBL-SCID mice leads to theprimary consequence of HIV-1 infection of humans,

loss of CD4+ T cells.8,9 All patient and laboratoryisolates of HIV-1 and HIV-2 tested to date have beeninfectious in the hu-PBL-SCID model; this includesthree HIV-2 isolates, and HIV-1 clade B, D and Eisolates. Individual HIV-1 isolates differ in the effi-ciency of primary infection, the rate of viral replica-tion, and the rate of CD4+ T-cell depletion.9,40,41 Acomparison of many of the viruses we have studied ispresented in Figure 1, which shows rate of CD4+ T-celldepletion plotted versus replication rate and cyto-pathic effects (rapid cell killing, syncytial induction).The rate of CD4+ T-cell loss varies from very rapid(e.g. isolate 89.6 which causes total loss of CD4+ Tcells within 2 weeks of infection) to very slow (lessthan 25% reduction in CD4+ T cells at 4 weeks afterinfection). Few viruses establish infection withoutcausing CD4+ T-cell depletion. Two HIV-2 isolates, aswell as HIV-1SF2∆nef (nef accessory gene depletion)and the Mu1 envelope mutation42,43 fall into thiscategory. Although the HIV-2 isolates replicate well inthe hu-PBL-SCID model, the two mutated HIV-1isolates show low infectivity and replication.

Several findings are evident in the data presented inFigure 1. First, there is considerable variation between

Figure 1. The relationship between extent of CD4+ T-celldepletion caused by HIV infection of hu-PBL-SCID mice (Xaxis), the in-vivo replication rate of the virus isolate (Y axis),and the cell tropism and in-vitro cytopathic effects of thevirus (Z axis). Each of the virus isolates (designated on thecolumn) was employed in at least two replicate experimentswith five mice per infected group, and some of the viruses(SF162, SF33, NL4-3, 89.6) have been used in 5–10experiments. Overlapping columns indicate no differencesbetween the three viruses studied. There is a tendency forviruses with high replication and high cytopathicity to causerapid CD4+ T-cell depletion, but some notable exceptionsto this trend exist (e.g. HIV1 SF33). Deletion of the nefgene seems to have a more deleterious effect on a lesspathogenic isolate like SF2 than on a highly pathogenic onelike 89.6.

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different viral isolates in the hu-PBL-SCID model,which reflects the biologic heterogeneity of HIV-1 ingeneral.44 Second, there is a correlation betweenreplication rate and extent of CD4+ T-cell depletion,although several of the nef deletion mutants and theHIV-1SF33 isolate45 are exceptions to this generaltrend. Deletion of the nef gene slows CD4+ T-celldepletion, but does not prevent it except in the case ofthe poorly infectious SF2 mutant. These results aresimilar to those seen with nef deletion mutants ofSIV46 and those reported with nef deletion mutants inthe SCID-hu model.47 Thus, use of the hu-PBL-SCIDmodel yields a clear phenotype for nef deletionmutants as do other animal models. In contrast, thein-vitro phenotype of nef deletion mutants is oftensubtle, and has led to conflicting interpretations48-50

of nef function (hence its misnomer, negative effectfactor). Third, several non-cytopathic, macrophage-tropic HIV-1 and HIV-2 (UC1) isolates cause rapid lossof CD4+ T cells, so there is no consistent relationshipbetween in-vitro cytopathic effects and in-vivo patho-genicity.9 Patient isolates from rapid progressors[WEAU, 89.651,52] do cause very rapid loss of CD4+ Tcells in hu-PBL-SCID mice, however. We are thus inagreement with Kaneshima et al 53 that highly cyto-pathic isolates from rapid progressors often (but notalways; e.g. SF33) show a more severe phenotype inSCID-hu or hu-PBL-SCID mice. The other findingpresented in Figure 1 is that deletion of the viralaccessory genes vpr or vpu in the highly pathogenic89.6 HIV-1 isolate54 has no obvious impact on virusinfection and rate of CD4+ T-cell depletion in the hu-PBL-SCID model. This result is in agreement withrecently published findings of Aldrovandi et al 55 inthe SCID-hu thy/liver model.

Macrophage-tropism and HIV-1 transmissionefficiency

We have further investigated the basis of the surpris-ing rapid CD4+ T-cell depletion caused by infectionwith macrophage-tropic HIV-1 isolates. Macrophage-tropism refers to the ability of virus isolates to grow inprimary human macrophage/monocytes as well asprimary T cells but not in established T-cell lines ortumors. Cell tropism is determined by the gp120envelope gene.56-58 Analysis of recombinant virusesgenerated from HIV-1SF162 and HIV-1SF2

42,43,59 havemapped the rapid CD4+ T-cell depletion phenotypeto envelope, although mutations in the V3 loop hadvariable effects on viral replication and CD4+ T-cellloss in the hu-PBL-SCID model.41 Quantitative analy-

sis of viral load by proviral DNA copy number orplasma viral RNA content at different times afterinfection of hu-PBL-SCID mice with HIV-1SF162 andHIV-1SF33 has shown that mice infected with SF162show higher viral loads at days 7–9 after infection, butequal or lower virus burden at days 14–28 afterinfection [ref 9, and unpublished observations]. Themacrophage-tropic SF162 isolate thus appears to havean advantage in initial infection efficiency rather thanreplication rate when compared to SF33. Severaldifferent types of experiments suggest that macro-phage-tropic virus has a substantial advantage in theinitial cycle of virus replication in the hu-PBL-SCIDmodel. The experiments compared the macrophage-tropic SF162 isolate and the T-cell line-tropic SF33isolate (see Figure 1). First, the rate of CD4+ T-celldepletion caused by SF162 and SF33 differed sub-stantially if cell-free virus was used to infect mice, butthat difference disappeared if virus-infected cells wereused to transmit infection.40 Infected cells are knownto be highly efficient at transmitting virus.60 Second,cell-free virus stocks of SF33 prepared in mitogen-activated PBL or macrophage-depleted PBL wereindistinguishable in terms of infectious titer andability to cause CD4+ T-cell depletion in hu-PBL-SCIDmice. In contrast, SF162 prepared in the absence ofprimary macrophages was both less infectious thanvirus grown in unseparated PBL, and caused muchless CD4+ T-cell depletion in hu-PBL-SCID mice if thePBL graft had been macrophage-depleted prior toreconstitution of the SCID mice. These experimentssuggest that macrophage-tropic virus derived frominfected macrophages is much more easily trans-mitted than the same virus derived from infectedCD4+ T cells. However, virus derived from CD4+ Tcells can be pathogenic in the hu-PBL-SCID model ifit quickly finds a human macrophage/monocyte toinfect. These findings strongly suggest that macro-phage-derived HIV-1 is different than T-cell-derivedvirus. The structure of envelope,61,62 the level ofglycosylation,63-65 the state of oligomerization,66,67

and the acquisition of host cell membrane compo-nents68 could all contribute to these differences. Amodel highlighting these differences is presented inFigure 2.

Immunity to HIV-1 infection

One of the unique advantages of the hu-PBL-SCIDmodel is that the PBL graft can be derived fromrecently immunized donors, which permits the adop-tive transfer of an ongoing human immune response

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to the SCID mouse. This approach was used toevaluate resistance to HIV-1 infection in hu-PBL-SCIDmice derived from volunteers participating in HIV-1vaccine trials.69 Only a subset of donors had PBL thatwould adoptively transfer HIV-1 immunity to hu-PBL-SCID mice. These donors were initially immunizedwith recombinant gp160 envelope in a vacciniavector70 and were later reimmunized with recombi-nant gp160 alone.71 If PBL were collected within sixmonths of the last immunization, they were able toprotect hu-PBL-SCID mice against challenge with thevaccine strain of virus.69 Interestingly, the protectiveeffect correlated better with T-cell immune status thanwith neutralizing antibody in the donor. These resultsstimulated two studies of passive transfer of immunityto HIV-1 with cytotoxic T-lymphocyte (CTL) clones15

or neutralizing antibody.16 Transfer of high numbersof CTL was protective against HIV-1 challenge if CTLinjection preceded virus challenge. However, theprotective effect appeared to have two components:an HLA-restricted, cytotoxic effect and an antiviralactivity mediated by any activated CD8+ T-cell clone.

This finding suggests that the CD8+ T-cell antiviraleffect that is prominent in vitro72-74 can also bedetected in the hu-PBL-SCID animal model, and maybe an important component of cellular resistance toviral spread in humans. In a second study,16 therecombinant b12-IgG1 neutralizing antibody75,76 wasinjected into hu-PBL-SCID mice prior to challengewith the SF2 isolate of HIV-1. The antibody showedcomplete protection of the mice at doses between 3–7mg/kg. Substantial levels of neutralizing antibodythus appear to be required for sterilizing immunity,although lower amounts might slow virus spread.Koup and colleagues17,77 have performed similarstudies which are reviewed elsewhere in this volume.

Epstein-Barr virus and human B-celllymphoproliferative disease in hu-PBL-SCIDmice

The introduction of PBL from donors infected withEpstein-Barr virus (EBV) into SCID mice leads to ahigh incidence of EBV-associated lymphoproliferative

Figure 2. HIV produced by human macrophages is different from the same virus produced by CD4T cells. These differences are primarily in the envelope gp120 molecule, and perhaps acquired cellmembrane components. The CD4+ T-cell-derived virion is shown in the standard cartoon withsingle gp120 monomers displayed on the membrane [adapted from Greene, WC, ref 87 withAuthor’s permission], whereas the macrophage-derived virion has trimers of gp120 that have beendifferentially glycosylated and have different antibody-exposed epitopes. The small squiggly lineson the T-cell-derived virion represent host components, while the extensive globular surfacemolecules adjacent to the gp120 trimers represent much more extensive representation ofmacrophage host components. The most easily detected host components are MHC molecules, butother important surface components could be passively acquired and lead to altered infectivity(e.g. LFA-1, ICAM-1, other integrins, CD14, etc.).

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disease similar to that seen in immunosuppressedorgan transplant recipients78,79 or in late stageAIDS25,26 The use of the hu-PBL-SCID model hasgiven considerable insight into how this process mighttake place. A previously described23 model for thepathogenesis of EBV-driven lymphoproliferative dis-ease is presented in Figure 3. Activation of CD4+

T-cell lymphocytes is important in initiating theprocess. CD4 T cells are required for lymphoprolifer-ative disease (ref 80, our unpublished observations)and administration of anti-CD40 ligand (CD40L)antibodies blocks appearance of tumors (M. Eckert, I.Atencio, D. Mosier, unpublished observations). How-ever, both of these manipulations substantiallydecrease the success of the PBL reconstitution, andonly a few B lymphocytes survive two weeks afterinjection. Nonetheless, anti-CD40L treatment doesnot totally block tumor induction. We have observed areduction to 33% tumor incidence in hu-PBL-SCIDmice treated with anti-CD40L29 for the first six weeksafter PBL reconstitution. The latent period untiltumor detection increased from 35 ± 3 days to 56–274days. Some small number of human B cells mustsurvive without activated T-cell help for at least sixweeks, and these cells can eventually give rise to EBV-driven B-cell lymphoproliferative disease. A secondline of evidence that T-cell-dependent B-cell differ-entiation is a critical event in tumor formation is therestriction of lytic cycle EBV replication to plasmacy-

toid cells expressing high levels of CD38 and lowerlevels of CD23.24,81 The lymphoblastoid componentof hu-PBL-SCID tumors harbors EBV that is tightlylatent.24 The production of infectious EBV by plasma-cytoid cells may lead to the infection and transforma-tion of new B cells, thus establishing a self-renewingcycle of transformation.

There is heterogeneity in the ability of PBL fromEBV-positive donors to give rise to tumors.22,82 Severalfactors seem to contribute to the variable outcome.One is likely to be the strength of the CD8+ T-cellresponse to EBV and how well it is maintained inSCID mice. Rapid loss of control of EBV-infected cellsmay set up a situation analogous to immune suppres-sion associated with post-transplant lymphomas. Sec-ondary genetic events in individual B cells may add totheir tumorogenic potential.82,83 Variations in EBVfrom different patients may lead to different trans-forming potential in the hu-PBL-SCID model.84 Cer-tain EBV-positive donors whose PBL rarely give rise totumors clearly have EBV with low transforming activity(G. Picchio, R. Rochford, D. Mosier; unpublishedobservations). B cells infected with EBV with deletionsin the critical EBNA-2 gene are attenuated for tumorformation in hu-PBL-SCID mice.85 Finally, the naturalkiller cells present in SCID mice may vary in theirability to control early tumors. Tumor latency isshortened in hu-PBL-SCID beige mice,86 where thebeige mutation introduces impaired NK cell lysis. As

Figure 3. Hypothetical scheme of B-cell transformation caused by EBV activation in hu-PBL-SCIDmice23. CD4+ human T lymphocytes are activated in the SCID environment. They provide cognatehelp (see review by Ifversen and Borrebaeck, this issue) to resting B lymphocytes, a small numberof which harbor latent EBV episomes. This T-dependent B-cell activation leads to thedifferentiation of the B cell to a plasma cell, at which time cell division stops and the EBV lytic cycleis activated. The plasma cell then produces infectious EBV which can infect and transform otherB cells, with the virus remaining latent until another round of plasma cell differentiation takesplace.

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detailed by Amadori et al elsewhere in this issue, thehuman cytokine profile may impact the incidence oflymphoproliferative disease. We have noted that PBLfrom atopic individuals are more likely to causeantibody-mediated graft-versus-host disease (anti-platelet and anti-mouse RBC antibodies) in hu-PBL-SCID mice, so the fate of the B-cell graft may be quitedifferent depending on the human donor. Finally, wehave noted differences in IL-10 and IL-6 levels intumors derived from different donors (R. Rochford,G. Picchio, D. Mosier; unpublished observations),suggesting that intrinsic cytokine production could berelated to tumor progression. Despite these variables,the study of the interaction of EBV and differentiatinghuman B lymphocytes in the hu-PBL-SCID modelremains a fertile area of investigation.

Acknowledgements

The work cited in this review is the result of experimentsperformed by several individuals, including R. Gulizia, G.Picchio, R. Rochford, B. Torbett, R. Van Kuyk, P. Parren, J.Glynn, Y. Ling, M. Eckert and I. Atencio. Many collaboratorshave supplied virus isolates and provided helpful discussion.These include J. Levy, D. Trono, R. Collman, G. Shaw and H.Kestler. This work was partially supported by NIH grantsAI29182 and CA65391, the Leukemia Society (R.R.), andthe University of California AIDS Research Program (J.G.).This is publication number 10085-IMM from The ScrippsResearch Institute.

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