Conserved Between Human and Mouse with CCR5 and Its

Home , CCR5, CCR8 (gene), CXCR4

The Primate Lentiviral Receptor Bonzo/STRL33 Is Coordinately Regulated with CCR5 and Its Expression Pattern Is Conserved Between Human and Mouse This information is current as of September 27, 2021. Derya Unutmaz, Wenkai Xiang, Mary Jean Sunshine, Jim Campbell, Eugene Butcher and Dan R. Littman J Immunol 2000; 165:3284-3292; ; doi: 10.4049/jimmunol.165.6.3284 http://www.jimmunol.org/content/165/6/3284 Downloaded from

References This article cites 58 articles, 30 of which you can access for free at:

http://www.jimmunol.org/content/165/6/3284.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Primate Lentiviral Receptor Bonzo/STRL33 Is Coordinately Regulated with CCR5 and Its Expression Pattern Is Conserved Between Human and Mouse1

Derya Unutmaz,2* Wenkai Xiang,* Mary Jean Sunshine,*† Jim Campbell,‡ Eugene Butcher,‡ and Dan R. Littman3*†

Chemokines play necessary and important roles in regulating the trafficking of lymphocytes to intra- or interlymphoid tissues as well as to sites of inflammation. The complex migratory patterns of lymphoid lineage cells is governed by subset-specific expression of chemokine receptors and their access to specific ligands. Several chemokine receptors and chemokine receptor-like orphan receptors also serve, in conjunction with CD4, as coreceptors for infection by human and simian immunodeficiency viruses (HIV and SIV). Here we show that the expression pattern of Bonzo/STRL33, an orphan SIV/HIV coreceptor, is highly restricted to the Downloaded from memory subset of T cells and is up-regulated upon stimulation of these cells with IL-2 or IL-15. Both the pattern and the regulation of Bonzo expression closely paralleled that of CC family chemokine receptors CCR5 or CCR6 and inversely correlated with CXCR4 expression. However, in striking contrast to CCR5, Bonzo expression was not down-modulated by PMA or mitogen stimulation of T cells. Targeted replacement of the Bonzo gene with a gene encoding green fluorescent protein in mice revealed that the expression and cytokine regulation of mouse Bonzo are comparable to those of its human counterpart. The similar expression and regulation patterns of Bonzo and the HIV coreceptor CCR5 may have implications for understanding the role of http://www.jimmunol.org/ HIV/SIV receptors in viral evolution and pathogenesis. The Journal of Immunology, 2000, 165: 3284–3292.

hemokine receptors belong to a subset within the super- referred as T-tropic) of HIV-1 (11–16). Additionally, CCR2, family of seven-transmembrane domain, G protein-cou- CCR3, CCR8 and CX3CR1 have been reported to be used by some C pled receptors that generally function to direct the com- of the HIV/SIV isolates, albeit at lower efﬁciencies (17–19). Sev- plex migratory or trafﬁcking patterns of leukocytes (1, 2). Most eral chemokine-receptor-like orphan receptors have also been chemokines are small secreted proteins that are grouped according shown to function as coreceptors for HIV and SIV strains (20–23). to the highly conserved position of cysteine residues within their One of these seven-transmembrane domain orphan receptors,

N-terminal region into C, CC, CXC, and CX3C families (3–5). Bonzo (also named STRL33 or TYMSTR), was identified as a by guest on September 27, 2021 Although chemokine receptors often exhibit multiple ligand spec- principal coreceptor for several strains of SIV (including SIVagm ificities, this promiscuity is generally limited to the binding of and SIVsm family viruses) as well as some HIV-2 and HIV-1 chemokines within the same family (2). Because of this, chemo- strains (20–23). Bonzo is a putative chemokine receptor based on kine receptors are classified based on the family of chemokines sequence homology with other chemokine receptor family mem- that they bind. Greater sequence homology also exists within each bers. However, its biologic function is not understood, and no nat- of the CC and CXC families of chemokine receptors than between ural ligand has yet been identified among the known human che- the two families. Moreover, the CC chemokine receptors CCR1- mokines (21) (T. Schall, unpublished observation). Expression of

CCR5, CCR8, CCR9/10, and CX3CR1 are closely linked on chro- Bonzo mRNA is restricted to lymphoid tissues, PBMC, and pla- mosome 3p21 (6–10). centa (20–23). Interestingly, the Bonzo gene maps to human chro- Members of the chemokine receptor family also serve as core- mosome 3 close to the region where genes encoding CC family ceptors, in conjunction with the CD4 molecule, for entry of HIV chemokine receptors are clustered (21). Little is known about the and SIV into target cells (11–16). CCR5 is the major coreceptor for cell subset-specific expression and regulation of Bonzo. The in R5 strains (previously referred as M-tropic) of HIV-1 and most vivo distribution of Bonzo-expressing cells and the modulation of SIV strains, while CXCR4 allows entry of X4 strains (previously this expression may be important in determining the role of Bonzo in SIV and HIV pathogenesis. To address this issue we have used *Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, New a mAb specific for Bonzo to demonstrate that expression of this York University Medical Center, and †Howard Hughes Medical Institute, New York, receptor is highly restricted to the memory subset of resting NY 10016; and ‡Laboratory of Immunology and Vascular Biology, Department of human T cells. We further show that, similar to CCR5, Bonzo Pathology, Stanford University School of Medicine, Stanford, CA 94305 expression is up-regulated by the cytokines IL-2 and IL-15. How- Received for publication June 16, 2000. Accepted for publication June 28, 2000. ever, in contrast to CCR5, upon PMA treatment or TCR stimulation The costs of publication of this article were defrayed in part by the payment of page Bonzo was not down-modulated. A similar expression pattern was charges. This article must therefore be hereby marked advertisement in accordance ϩ with 18 U.S.C. Section 1734 solely to indicate this fact. observed on mouse CD4 T cells of mice in which a green fluores- 4 1 This work was supported by National Institutes of Health Grants AI33856 and cent protein (GFP) gene was knocked into the endogenous Bonzo AI36606 to D.R.L. locus. 2 Address correspondence and reprint requests to Dr. Derya Unutmaz, Department of Microbiology and Immunology, Vanderbilt University Medical School, AA-5216 Medical Center North, Nashville, TN 37215. E-mail address: derya.unutmaz@ 4 Abbreviations used in this paper: GFP, green fluorescent protein; MIP, macrophage mcmail.vanderbilt.edu inflammatory protein; SDF-1␣, stromal cell-derived factor; TC, tricolor; ORF, open 3 D.R.L. is an Investigator with the Howard Hughes Medical Institute. reading frame; ES, embryonic stem.

FIGURE 1. Bonzo Ab speciﬁcally stains transfected cells. a, HOS cells stably expressing Bonzo were generated by retroviral transduction of the Bonzo or CCR5 cDNA using the pBabe vector (20). Cells were stained with the anti-Bonzo mAb followed by a PE-conjugated goat-anti-mouse

Ab. b, 293T cells were transfected with a pcDNA3 plasmid encoding either http://www.jimmunol.org/ GFP or Bonzo-GFP fusion protein. Cells were then stained, 48 h post- transfection, with Bonzo Ab as described above. FIGURE 2. Expression of Bonzo on PBMC. Freshly isolated PBMC Materials and Methods were stained with anti-Bonzo mAb followed by the PE-conjugated sec- Generation of mAb against Bonzo ondary Ab and were subsequently stained with PE-conjugated Abs specific for other cell surface molecules. Three donors gave similar results, and data An mAb against Bonzo/STRL33 was raised at R&D Systems (Minne- from one donor are shown. Numbers in quadrants are percent positives. apolis, MN) by immunizing BALB/c mice with a syngeneic mouse Expression of Bonzo on a) T cells (CD3ϩ); b and c) CD4ϩ and CD8ϩ T myeloma (NSO) transfected with full-length human Bonzo/STRL-33 cell subsets; d) memory (CD4ROϩ) and naive (CD45ROϪ) T cells (gated by guest on September 27, 2021 with a polyhistidine fused to the C-terminal end of the receptor for CD3ϩ cells); e) NK cells (CD16ϩ); f) ␥␦ T cells (gated on CD3ϩ cells); sequence. The polyhis serves as an epitope tag that can be used to identify ϩ ϩ g) B cells (CD19 ); h, monocyte expression (CD14 ); and i) dendritic (via intracellular staining in FACS or via Western blot) transfected clones that appear to be expressing high levels of the gene of interest. An immu- cells. The dendritic cells were identified as follows: PBMC were first nization protocol (24) for soluble protein was adapted for use with whole stained with anti-Bonzo Ab as described above and then with FITC-con- cells as the immunogen. The priming immunization was performed by jugated anti-CD3, -CD14, and -CD19 and TC-conjugated anti-HLA-DR mixing the cell suspension in PBS with an equal volume of emulsified Abs. A gate was set to exclude FITC-positive cells (T cells, B cells, and MPL/TDM adjuvant (Ribi); subsequent boosts used cells in PBS alone. monocytes). The remaining HLA-DRϩ cells identify DC. Following immunization, lymph node cells were used for polyethylene glycol-mediated fusion following conventional protocols. After 7 days of culture, supernatants were screened for Abs that could bind to paraform- aldehyde-fixed NSO/STRL-33/polyHis cells used for immunization. Cul- penicillin (50 U/ml; Life Technologies), streptomycin (50 ␮g/ml), sodium tures that were positive in this primary screen were then tested for binding pyruvate (1 mM; Life Technologies), and glutamine (2 mM; Life Tech- to NSO cells that had been transfected with an irrelevant gene (GDF-9) nologies). T cell lines were prepared by activation of purified resting T also expressed as a polyhis construct. One clone was chosen and subcloned cells with allogeneic PBMC and were treated with 50 ␮g/ml mitomycin C based on strong binding of its supernatant to the unfixed relevant trans- (Sigma) for 30 min at 37°C and 5 ␮g/ml PHA (Sigma). Cells were split 3 fectants. This hybridoma secretes an IgG2b ␬ mAb that was purified and days postactivation, expanded, and maintained in culture medium supple- used in subsequent experiments. This Ab is designated MAB699. mented with 200 U/ml recombinant IL-2 (Chiron). T cell lines were maintained by restimulating the cells every 2 wk with PHA and mitomycin Preparation of human PBMC and resting T cells C-treated allogeneic PBMCs. Culture of T cells with cytokines has been PBMC were separated from buffy coats of healthy donors (New York previously described (26, 27). Cytokines IFN-␥, IL-4, IL-7, IL-12, and Blood Bank, New York, NY) through Ficoll-Hypaque (Pharmacia, Upp- IL-15 and chemokines RANTES, macrophage inflammatory protein-1␣ sala, Sweden). Purification of T cells was performed as previously de- (MIP-1␣), MIP-1␤, and stromal cell-derived factor 1␣ (SDF-1␣) were all scribed (25). Briefly, monocytes were first removed from PBMC by plastic obtained from R&D Systems. adherence for2hat37°C. Nonadherent cells were incubated with anti-CD4 or anti-CD8 conjugated with Dynabeads (Dynal, Oslo, Norway) at a 1:4 Ab staining and FACS analyses target/bead ratio. The bead-bound cells were recovered using a magnet Cells were incubated with the relevant Ab on ice for 30 min in PBS buffer (Dynal) washed at least four times to remove unbound cells. The CD4ϩ or ϩ with 2% FCS and 0.1% sodium azide. For staining of Bonzo, cells were CD8 cells were detached from the beads using Detachabead according to incubated with anti-Bonzo mAb at 3 ␮g/ml; after two washes, cells were the manufacturer’s instructions (Dynal). These cells were then incubated incubated with goat anti-mouse IgG conjugated to PE or tricolor (TC) with anti-HLA-DR Ab followed by Dynabeads conjugated with goat anti- (Caltag, South San Francisco, CA). The cells were washed twice again and mouse IgG for magnetic removal of preactivated T cells and contaminating blocked with excess mouse IgG (100 ␮g/ml) followed by staining with dendritic cells or macrophages. directly conjugated Abs against the relevant cell surface molecules. The Media, reagents, and T cell cultures Abs used for staining were PE, FITC, TC, or peridin chlorophyll protein conjugates of anti-human CCR5 and CXCR4; anti-mouse CD3, CD4, CD8, The culture medium used in all experiments was RPMI (Life Technologies, CD44, CD45RB, and ␥␦TCR (PharMingen, San Diego, CA); anti-human Grand Island, NY) supplemented with 10% FCS (HyClone, Logan, UT), CD4 and CD45RO, and secondary Abs goat anti-mouse PE or FITC 3286 REGULATION AND EXPRESSION PATTERN OF BONZO//STRL33

DNase I (Roche, Indianapolis, IN). RNA (1 ␮g) was used in an Access RT-PCR system (Promega, Madison, WI) with 50 pmol of sense (GCT TGC TCA TTT GGG TG) and antisense (CGC CGC GTC GAC CTT CTC TAA GTG TGG CAA GGC) Bonzo primers. RT was performed for 45 min at 48°C, and cDNA amplification was conducted for 40 cycles at 94°C for 30 s, 58°C for 1 min, and 68°C for 2 min. To exclude possible contami- nation with genomic DNA, control reactions in which reverse transcriptase was omitted were performed in parallel. Fluorescence microscopy Mouse organs were fixed in 4% formaldehyde for at least 12 h and incubated in 15% sucrose in PBS for 1 h. The samples were embedded in HistoPrep (Fisher, Fairlawn, NJ) and later frozen with dry ice. Samples were cut into 15-␮m sections with a cryostat. Fluorescence microscopy was used to visualize GFP expression. Results Bonzo expression pattern on PBMC An mAb reacting against human Bonzo was obtained as described in Materials and Methods. This Ab binds specifically to HOS cells that stably express the Bonzo cDNA (Fig. 1a) (20). Similar results Downloaded from were obtained using Bonzo-transduced NIH-3T3 cells or a mouse thymoma line (data not shown). In addition, 293T cells were transfected with a plasmid encoding Bonzo-GFP fusion and stained FIGURE 3. Bonzo and CCR5 are expressed on similar T cell subsets. with anti-Bonzo Ab. Only Bonzo-GFP-expressing cells were pos- PBMC were stained with mAbs against either Bonzo or CCR5 and then itively stained with anti-Bonzo mAb, and this correlated with GFP with secondary PE-conjugated Ab followed by CD3-TC and FITC-conju- expression. To exclude the cross-reactivity of the Ab with other http://www.jimmunol.org/ ϩ gated anti-CD8, -CD4, or -CD45RO mAbs. Gating was restricted to CD3 chemokine receptors, we also stained HOS cell lines stably ex- cells. a, CCR5 expression on T cell subsets and comparative staining of pressing CCR1, CCR3, CCR4, CCR7, CCR8, APJ, and V28. None Bonzo (bottom panel). b, Bonzo expression of T cell subsets from a CCR5- of these cell lines was stained with anti-Bonzo mAb (data not null individual. shown). We then determined the expression pattern of Bonzo on PBMC of normal donors using multicolor FACS analysis. Expres- (Caltag); anti-human CD3, CD8, CD14, CD16, CD19, CD45RA, HLA- ϩ DR, and ␥␦TCR (all from Becton Dickinson, Palo Alto, CA); or anti- sion of Bonzo was found to segregate primarily to CD3 T cells ϩ ϩ human CCR6 (R&D Systems). Staining was analyzed on a FACScan using (Fig. 2a). Both CD4 and CD8 T cell subsets expressed Bonzo CellQuest software (Becton Dickinson). Live cells were gated based on (Fig. 2, b and c), although the percentage of Bonzoϩ cells was forward and side scatter. Intracellular staining was performed using Cyt- by guest on September 27, 2021 ofic/Cytoperm solution according to the manufacturer’s protocol (Phar- Mingen). To perform FACS analyses on fresh human thymocytes, thymi were obtained from 7- to 9-mo-old pediatric heart surgery cases. Thymic tissue was disrupted by mincing and forcing through stainless steel mesh. Thymocytes were incubated twice at 37°C for 30 min in complete medium to remove adherent cells. Single-cell suspensions were then placed on ice and stained for FACS analysis as described above. Gene targeting in embryonic stem cells and generation of mice A 129/Sv mouse genomic DNA library was screened with human Bonzo cDNA and a 16-kb DNA fragment was isolated. Sequence analysis identified a 1.2-kb intronless open reading frame (ORF) homologous to that of the human Bonzo gene. To generate an EGFP (Clontech) knockin targeting vector, three DNA fragments were sequentially inserted into a pBS-KSϩ plasmid: 1) a GFP expressing cassette followed by SV40 poly(A)n sequence, 2) a neomycin resistance cassette (neoR) flanked with loxP sites (28), and 3) a HSV thymidine kinase cassette (HSV-TK). Subsequently, an 8-kb ApaI-NotI genomic fragment downstream of the Bonzo ORF was inserted between the neoR and HSV-TK cassettes, whereas a 1.6-kb genomic fragment upstream of the Bonzo ORF and containing the 5Ј-un- translated region was inserted 5Ј of the GFP ORF. The resulting targeting vector was linearized with ClaI and electroporated into 129/Sv-derived E14 embryonic stem (ES) cells. G418-resistant ES cell clones were then screened for homologous recombination. To eliminate possible interfer- ence from the neo gene, correctly targeted clones were electroporated with a Cre recombinase-expressing vector (pCMV-Cre) to delete the neoR cassette, which was confirmed by sensitivity to G418 and by PCR or Southern blotting for the absence of the neoR-coding region. The resulting clones were microinjected into C57BL/6 blastocysts. Chimeric mice were mated with wild-type C57BL/6 mice to produce heterozygous progeny. Six- to 8-wk-old littermates from the mating of heterozygous mice were then analyzed. FIGURE 4. Coexpression of Bonzo with CCR5. PBMC were stained RT-PCR analysis with Bonzo as previously described, followed by PE-conjugated anti- Total RNA was extracted from mouse lymph nodes and spleen using Trizol CCR5, -CCR6, or -CXCR4 and TC-conjugated anti-CD3 mAbs. Profiles reagent (Life Technologies). The RNA was further treated with RNase-free are gated on CD3ϩ cells. The Journal of Immunology 3287

FIGURE 5. Cytokine-mediated regulation of Bonzo and CCR5 on resting CD4ϩ and CD8ϩ T cells. Purified CD4ϩ or CD8ϩ resting T cells were cultured for 8 days in the presence of the cytokines IL-2 (200 U/ml), IL-4 (20 ng/ml), IL-7 (10 ng/ml), and IL-15 (10 ng/ml). Cells were then stained with mAbs specific for Bonzo and for CD45RO and CD4 or CD8 (top and lower panels, respectively). Downloaded from always higher within the CD8ϩ subset in samples obtained from CXCR4 was inversely correlated (Fig. 4). This inverse expression different donors (data not shown). T cells can also be subdivided pattern reflects lower levels of CXCR4 on memory T cells (30) into memory and naive subsets based on expression of CD45 iso- (data not shown), which preferentially express Bonzo (Fig. 2d). forms, CD45ROϩRAϪ and CD45ROϪRAϩ, respectively (29). We also analyzed the expression of Bonzo within the thymus Bonzo was exclusively expressed on CD45ROϩ T cells (Fig. 2d). and secondary lymphoid organs, adenoids and tonsils. Bonzo,

About 30–40% of ␥␦ T cells also expressed Bonzo (Fig. 2f). The CCR5, and CCR6 expression was either undetectable or was http://www.jimmunol.org/ few CD3-negative cells that expressed Bonzo were CD16ϩ NK present on Ͻ1% of thymocytes, whereas the majority of the thy- cells (Fig. 2e), and no expression was detected on B cells, mono- mocytes expressed CXCR4 (data not shown). Expression profiles cytes, or dendritic cells (Fig. 2, g, h, and i, respectively). of Bonzo, CCR5, and CCR6 on the lymphocytes isolated from It has been reported that expression of some chemokine recep- tonsils or adenoids were similar to those observed on PBMC, al- tors, such as the major HIV coreceptor CCR5, is also restricted to though a greater proportion of T cells was positive, and most of the memory subset of T cells (30). As shown in Fig. 3, the ex- these cells expressed CD45ROϩ as well as the early activation pression profile of CCR5 on resting T cells very closely parallels marker CD69 (data not shown). that of Bonzo. Indeed, costaining of PBMC with Abs specific for Bonzo and CCR5 showed that most Bonzo-positive cells were also by guest on September 27, 2021 CCR5ϩ (Fig. 4). PBMC from a healthy donor with a mutation in Regulation of Bonzo expression by cytokine stimulation of the CCR5 gene that prevents its cell surface expression (31) ex- resting T cells hibited normal expression of Bonzo (Fig. 3b), demonstrating that The concordant subset distribution of Bonzo and CCR5 suggested absence of CCR5 from the cell surface does not negatively influ- that the surface expression of these molecules may be regulated by ence Bonzo expression. Most Bonzo-positive resting T cells also similar mechanisms. It has been noted that IL-2 or IL-15 stimula- expressed CCR6 (Fig. 4), whereas expression of Bonzo and tion of human T cells can up-regulate some of the CC chemokine

FIGURE 6. Cytokine-mediated coregulation of Bonzo and chemokine receptors CCR5, CCR6, or CXCR4 on resting T cells. Puriﬁed CD4ϩ T cells were cultured with cytokines as described in Fig. 5. Cells were stained for Bonzo, CCR5, CCR6, or CXCR4 expression as described in Fig. 4. 3288 REGULATION AND EXPRESSION PATTERN OF BONZO//STRL33

freshly isolated PBMC. Little expression was observed on CD4ϩ T cells cultured with IL-4 or IL-7 (Fig. 5), and none was found with IL-6, IL-12, or IFN-␥ (data not shown). However, significant up-regulation of Bonzo was observed on CD8ϩ T cells cultured with IL-7 (Fig. 5, lower panel). IL-2 and IL-15 also up-regulated CCR5 and CCR6 expression on CD4ϩ T cells, and most Bonzo- expressing cells also coexpressed these CC chemokine receptors (Fig. 6). Similar results were obtained with CD8ϩ T cells (data not shown). This result recapitulates the coordinate expression profile of Bonzo with CCR5 or CCR6 in freshly isolated PBMC (Fig. 4). Conversely, Bonzo-positive cells had slightly lower CXCR4 expression than Bonzo-negative cells after stimulation with IL-2 or IL-15 (Fig. 6). CXCR4 expression was significantly up-regulated in the presence of IL-4, and there was also a moderate increase in response to IL-2 or IL-15, in accordance with previous reports (35–37). Post-transcriptional regulation of Bonzo cell surface expression

Most chemokine receptors contain a DRY sequence motif that ap- Downloaded from pears to be required for coupling to G proteins (38). In contrast to other CC and CXC-family chemokine receptors, Bonzo possesses a noncanonic DRY box sequence at the second intracellular loop. This raised the possibility that in response to its physiologic ligand Bonzo may not signal in a similar fashion to the other chemokine

receptors. Recently, it has been reported that chemokine receptors http://www.jimmunol.org/ are down-regulated through ligand-mediated endocytosis (39–42). Therefore, we reasoned that if any of the CCR5 ligands also bound to Bonzo, we may be able to detect this through down-regulation of Bonzo in the absence of detectable signaling. Fig. 7a shows that none of the known CCR5 ligands (MIP-1␣, MIP-1␤, and RANTES) had any effect on Bonzo expression, whereas they completely down-modulated CCR5 at the same concentrations. As expected, CXCR4 was down-modulated by its ligand SDF-1␣, but not by CCR5 ligands (Fig. 7a). Interestingly, stimulation of cells by guest on September 27, 2021 with phorbol ester (PMA) also did not have any effect on Bonzo expression, although CCR5 and CXCR4 expression was completely down-modulated (Fig. 7b). Similarly, upon restimulation of T cell lines with mitogen, CCR5 expression was down-modulated, but that of Bonzo was unaffected (Fig. 7c). To determine whether Bonzo is premade, stored in intracellular compartments, and re- leased to cell surface upon signaling, we performed intracellular staining on resting or cytokine-stimulated primary T cells with anti-Bonzo Ab. No difference was observed between cell surface FIGURE 7. Effects of chemokines, PMA, and mitogen stimulation on staining alone or in conjunction with intracellular staining (data Bonzo, CCR5, and CXCR4 cell surface expression. To maximize expres- not shown). sion levels, CD4ϩ cells cultured with IL-15 for 12 days were used in the Targeted disruption and knockin of EGFP into the mouse Bonzo Bonzo and CCR5 down-regulation experiments, and IL-4-cultured cells were used for examining CXCR4 down-regulation. a, Cells were cultured gene locus for 24 h in the presence of the chemokines RANTES, MIP-1␣, MIP-1␤,or To gain insight into the expression pattern and function of Bonzo SDF-1␣ (1 ␮g/ml each) before staining. b, Cells were stimulated with 15 in mice, we used a gene-targeting strategy that replaced the Bonzo- ng/ml PMA for 12 h and stained for chemokine receptor expression. c, ϩ coding sequence with that of EGFP (Fig. 8A) Germline transmis- Activated CD4 human T cell lines maintained in IL-2 were stained for sion was obtained with two independent ES cell clones. Deletion chemokine receptor expression 2 wk poststimulation (upper panel)or of the Bonzo gene was verified by Southern blot analysis (Fig. 8B). 3days after restimulation through TCR (lower panel). In an RT-PCR assay, a 3Ј portion of Bonzo mRNA was amplified from total RNA derived from lymph node and spleen of both Bonzoϩ/Ϫ (Bzϩ/Ϫ) and BonzoϪ/Ϫ (BzϪ/Ϫ) mice. No amplification receptors (27, 32–34). We asked whether Bonzo is similarly reg- was observed from BzϪ/Ϫ mice (Fig. 8C). Mating of heterozygous ulated by cytokines on resting human T cells. CD4ϩ and CD8ϩ mice gave rise to BzϪ/Ϫ mice in Mendelian proportions. BzϪ/Ϫ resting T cells were purified from PBMC and cultured for 8 days mice were phenotypically indistinguishable from Bzϩ/ϩ and Bzϩ/Ϫ in the presence of various cytokines. Culture of CD4ϩ or CD8ϩ T littermates in a specific pathogen-free environment. Histologic cells with IL-2 or IL-15 resulted in up-regulated expression of analysis of organs displayed no morphologic difference among the Bonzo exclusively on CD45ROϩ memory T cells (Fig. 5), with three genotypes. Flow cytometric analysis of cells from lymphoid expression on CD8ϩ T cells usually greater than that on CD4ϩ T organs showed no alteration in various cell populations (data not cells. This result is consistent with the staining pattern observed on shown). Proliferation of T cells from Bzϩ/Ϫ or BzϪ/Ϫ mice was The Journal of Immunology 3289

FIGURE 8. Targeted knockin of EGFP in the mouse Bonzo gene. A, From the top, the wild-type Bonzo genomic structure, the targeting construct, and Downloaded from the targeted Bonzo/EGFP knockin structure, respectively. The arrows above each gene depict the directions of transcription. ‚, loxP sites. H, HindIII; A, ApaI; RV, EcoRI; C, ClaI. B, Southern blot analysis of progeny of heterozygous intercrossing. The probe used is depicted by the solid bar beneath the targeted structure in A. The wild-type allele yields a 7-kb EcoRV genomic fragment, whereas the targeted allele yields a 6.6-kb fragment. C, RT-PCR analysis of expression of Bonzo mRNA in mutant mice. The primers used are described in Materials and Methods. In a TAE gel, a 590-bp DNA fragment is amplified from wide-type (top panel, lane 2) and heterozygous (lane 3) mice, but is absent in BzϪ/Ϫ mice (lane 4). A similar RT-PCR assay was conducted for GADPH mRNA, as a loading control (lower panel). http://www.jimmunol.org/ assessed in response to different concentrations of anti-CD3 Ab in 10d), suggesting that, as in human CD4ϩ T cells, Bonzo is pri- the presence or the absence of anti-CD28 Ab. No difference was marily expressed in the memory subset of mouse CD4ϩ T cells. observed between BzϪ/ϩ and BzϪ/Ϫ cells (data not shown). Nu- Finally we determined whether, similar to human Bonzo, ex- cleoprotein-keyhole limpet hemocyanin-immunized Bzϩ/Ϫ and pression of mouse Bonzo can be modulated by the presence of BzϪ/Ϫ mice also displayed similar levels of nucleoprotein-specific cytokines. Indeed, we observed dramatic up-regulation of GFP ex- IgM and IgG1 production (data not shown), suggesting that T cell pression after 10 days of culture with IL-2, but less with IL-7 and help and B cell function were normal in Bonzo-deficient mice. none with IL-4. These results suggest that cytokine-mediated reg- by guest on September 27, 2021 ulation of murine Bonzo follows the pattern of its human coun- Tracing mouse Bonzo expression in EGFP knockin mice terpart (Fig. 11). The in-frame substitution of EGFP for Bonzo yields a sensitive tool to trace Bonzo expression and regulation in vivo. Histologic Discussion analysis did not show any GFP expression in nonlymphoid tissues. The discovery that HIV and SIV use chemokine receptors to enter Strong green fluorescence was detected in spleen and lymphoid target cells raised hopes that chemokines and chemokine receptors nodes. In spleen, GFP-positive cells were clustered in the periar- might hold clues to understanding the progression of HIV-medi- teriolar lymphoid sheath area in the white pulp, where T cells ated pathogenesis. These discoveries also bolstered the general in- reside (Fig. 9). Notably, there was no difference in expression pat- terest in the regulation of lymphocyte migration by chemokine tern between Bzϩ/Ϫ and BzϪ/Ϫ mice. We next performed FACS analysis of GFP expression in different lymphoid subsets. GFP expression was undetectable in B cells, granulocytes, monocytes or macrophage, and dendritic cells (data not shown). In thymus, no expression was observed in CD4ϩCD8ϩ cells or double-negative thymocytes. Only a small subset of single-positive cells showed strong expression (data not shown). Within the lymph nodes and spleen almost all GFPϩ cells were CD3ϩ T cells (Fig. 10a). The mean fluorescence of GFP-expressing BzϪ/Ϫ cells was slightly higher than that of Bzϩ/Ϫ cells, most likely due to GFP expression from both targeted alleles (data not shown). Few CD4ϩ T cells expressed GFP, whereas the majority of CD8ϩ T cells were positive, with a broad distribution of inten- sity of GFP expression. We next analyzed the distribution of GFP expression on naive vs memory T cells in Bonzo knockin mice. CD8ϩ cells were all CD44ϩ, and it was difficult to discriminate naive vs memory cells (Fig. 10b). ␥␦ T cells from lymphoid organs or resident within the skin, reproductive tract, or gut also expressed FIGURE 9. Expression of GFP in spleen sections of Bonzo knockin ϩ GFP (Fig. 10c). Interestingly, GFP expression on CD4 murine T mice. The section shows GFP fluorescence in T cell-rich periarteriolar ϩ cells was restricted to the CD44 and CD45RBlow subset (Fig. lymphoid sheath area from Bonzo knockin mice. 3290 REGULATION AND EXPRESSION PATTERN OF BONZO//STRL33

FIGURE 11. Regulation of mouse Bonzo expression by cytokines. CD4ϩ T cells were puriﬁed from lymph nodes of Bzϩ/Ϫ and BzϪ/Ϫ mice. Cells (4 ϫ 105) were treated with cytokines (IL-2, IL-7, and IL-4) or activated by plate-bound CD3 Ab and assayed for GFP expression 3 and 10 Downloaded from days later. Most GFP-expressing cells were CD44high. The result is plotted as the percentage of CD4ϩ T cells that are GFP positive.

ϩ ϩ

Bonzo was expressed on CD4 , CD8 and ␥␦ subsets of T cells, http://www.jimmunol.org/ expression levels were usually higher on CD8ϩ and TCR␥␦ϩ T cells. Few NK cells were found to express Bonzo, and those that did had relatively low levels of CD16 expression (Fig. 2e), possi- bly representing recently activated NK cells. The coexpression of Bonzo and CCR5 on memory T cells is notable from the perspective of HIV coreceptor usage, because CCR5 is the major receptor used by most strains of HIV and almost all SIVs. Expression of CCR5 on the cell surface has been shown to be critical in transmission of HIV-1 infection, because a by guest on September 27, 2021 homozygous 32-bp deletion in the CCR5 gene prevents CCR5 expression and confers resistance against HIV-1 infection (31, 47, 48). We showed that T cells isolated from individuals who are homozygous for the CCR5⌬32 mutation expressed normal levels of Bonzo. Although Bonzo is a minor HIV-1 coreceptor, its role during transmission of infection and pathogenesis is unclear. How- FIGURE 10. GFP expression on lymphocyte subsets from Bonzo ever, Bonzo is a major SIV receptor and is also used by many knockin mice. FACS analysis of T cell subsets from lymph nodes of HIV-2 strains in vitro (18, 20). It is interesting to note that different Bonzo-GFP knockin mice was performed. a, GFP expression on CD3ϩ T species of nonhuman primates vary widely in their responses to cells; b, cells stained with anti-mouse CD8-TC and CD44-PE, gated on SIV infection, and people infected with HIV-2 usually have a more CD8ϩ T cells; c, IEL stained with PE-conjugated anti-TCR␥␦ Ab; d, GFP delayed progression to AIDS. It is possible that Bonzo may sub- expression on naive and memory mouse CD4 T cells. Lymph node cells stitute for CCR5 usage during infection with SIV and HIV-2 iso- stained with CD4-TC and with either CD44-PE or CD45RB-PE and ana- lates and perhaps influences the course of infection and pathogen- ϩ lyzed after gating was set on CD4 cells. esis. In contrast to CCR5, Bonzo was not expressed by macrophages or dendritic cells. This expression pattern may pre- clude its involvement during the initial phase of infection, where receptors. The recent development of Abs to chemokine receptors macrophage or dendritic cell infection is thought to be necessary has revealed the complex subset-specific expression of these re- for the virus to gain a foothold in the body (49). ceptors on T lymphocytes (43). In this report we demonstrate that Cytokine stimulation of T cells differentially regulates the sur- the expression pattern and regulation of the SIV/HIV coreceptor face expression of chemokine receptors. IL-2 has been described to and putative orphan chemokine receptor Bonzo strongly parallels potently up-regulate CCR1, CCR2, CCR5, CCR6, and CXCR3 those of CC family chemokine receptors. (27, 30, 32, 50, 51). More recently, IL-15 has been shown to have It has been shown that CC family chemokine receptors CCR2, similar effects on some of the CC chemokine receptors (27, 33). In CCR5, and CCR6 are expressed primarily on the CD45ROϩ or contrast, CXCR4 expression is dramatically up-regulated in the CD26ϩ memory subset of T cells (30, 44–46). The expression presence of IL-4 (35–37). We have shown that Bonzo, similar to pattern of Bonzo was similarly restricted to memory T cells. In this CC chemokine receptors, is induced through stimulation of highly regard we found that most Bonzo-positive cells also coexpressed purified resting T cells with IL-2 or IL-15 and is coexpressed with CCR5 or CCR6 on peripheral blood T cells. In contrast, Bonzo CCR5 or CCR6. The coordinated regulation of Bonzo and CCR5 was not coexpressed with high levels of CXCR4, reflecting lower on resting T cells by IL-2 or IL-15 recapitulates the ex vivo ex- CXCR4 expression on memory vs naive T cells (30). Although pression pattern. The Journal of Immunology 3291

Notably, the gene for Bonzo maps to chromosome 3, where the human thymus, Christie Doxsee for excellent technical help, and Dr. most of the chemokine receptors, especially those of the CC fam- Vineet N. Kewal-Ramani for many helpful suggestions, critique, and ily, are localized (21). Additionally, the amino acid sequence of reagents. Bonzo is similar to that of other members of the chemokine receptor family. Taken together with our results on its expression References pattern and cytokine-mediated regulation, these findings suggest 1. Murphy, P. M. 1996. Chemokine receptors: structure, function and role in mi- that Bonzo is also a chemokine receptor. However, Bonzo stands crobial pathogenesis. Cytokine Growth Factor Rev. 7:47. 2. Ward, S. G., K. Bacon, and J. Westwick. 1998. Chemokines and T lymphocytes: apart from other chemokine receptors, because its expression is more than an attraction. Immunity 9:1. resistant to PMA-induced down-modulation. This result contrasts 3. Schall, T. J., and K. B. Bacon. 1994. Chemokines, leukocyte trafficking, and with PMA-induced down-modulation of CXCR4 (52, 53), CCR5 inflammation. Curr. Opin. Immunol. 6:865. 4. Baggiolini, M., B. Dewald, and B. Moser. 1997. Human chemokines: an update. (Fig. 7b), or CCR6 (data not shown). However, Bonzo may still be Annu. Rev. Immunol. 15:675. down-modulated by its ligand(s), because the mechanisms of li- 5. Baggiolini, M. 1998. Chemokines and leukocyte traffic. Nature 392:565. gand- and PMA-induced down-modulation appear to be different 6. Raport, C. J., V. L. Schweickart, R. J. Eddy, T. B. Shows, and P. W. Gray. 1995. The orphan G-protein-coupled receptor-encoding gene V28 is closely related to (42, 52, 53). We did not detect any ligand-induced down-modu- genes for chemokine receptors and is expressed in lymphoid and neural tissues. lation of Bonzo in the presence of CCR5 ligands, but we cannot Gene 163:295. rule out that Bonzo is resistant to ligand-induced endocytosis and 7. Samson, M., P. Soularue, G. Vassart, and M. Parmentier. 1996. The genes encoding the human CC-chemokine receptors CC-CKR1 to CC-CKR5 (CMKBR1- thus may still be able to bind to these chemokines. CMKBR5) are clustered in the p21.3-p24 region of chromosome 3. Genomics Because there is no Ab yet available against mouse Bonzo, we 36:522. ␤ used the targeted knockin of GFP in place of the Bonzo gene as an 8. Daugherty, B. L., and M. S. Springer. 1997. The -chemokine receptor genes Downloaded from CCR1 (CMKBR1), CCR2 (CMKBR2), and CCR3 (CMKBR3) cluster within 285 indicator of Bonzo expression. We found that murine Bonzo is kb on human chromosome 3p21. Genomics 41:294. expressed in cell subsets similar to human Bonzo, namely in T 9. Bonini, J. A., S. K. Martin, F. Dralyuk, M. W. Roe, L. H. Philipson, and cells, particularly those displaying markers of effector/memory D. F. Steiner. 1997. Cloning, expression, and chromosomal mapping of a novel human CC-chemokine receptor (CCR10) that displays high-affinity binding for cells and those stimulated by IL-2 (data not shown). However, in MCP-1 and MCP-3. DNA Cell Biol. 16:1249. both mouse and human only a subset of memory cells expressed 10. Roos, R. S., M. Loetscher, D. F. Legler, I. Clark-Lewis, M. Baggiolini, and B. Moser. 1997. Identification of CCR8, the receptor for the human CC chemo-

Bonzo, similar to CCR5 expression. Chemokines regulate both the http://www.jimmunol.org/ kine I-309. J. Biol. Chem. 272:17251. inter- and intraorgan migration patterns of human T cell subsets. 11. Feng, Y., C. C. Broder, P. E. Kennedy, and E. A. Berger. 1996. HIV-1 entry Recently, human memory T cells were subdivided into two func- cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled tionally distinct subsets based on CCR7 expression (54). CCR7Ϫ receptor. Science 272:872. 12. Alkhatib, G., C. Combadiere, C. C. Broder, Y. Feng, P. E. Kennedy, memory cells express receptors for migration to inﬂamed tissues P. M. Murphy, and E. A. Berger. 1996. CC CKR5: a RANTES, MIP-1␣, MIP-1␤ and display immediate effector function. In contrast, CCR7ϩ mem- receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955. ory cells express lymph node-homing receptors and lack immedi- 13. Deng, H., R. Liu, W. Ellmeier, S. Choe, D. Unutmaz, M. Burkhart, P. Di Marzio, Ϫ S. Marmon, R. E. Sutton, C. M. Hill, et al. 1996. Identiﬁcation of a major co- ate effector function. Interestingly, the CCR7 cells were enriched receptor for primary isolates of HIV-1. Nature 381:661. for expression of CCR5, CCR6, and CCR1 (54). Based on the 14. Dragic, T., V. Litwin, G. P. Allaway, S. R. Martin, Y. Huang, K. A. Nagashima,

C. Cayanan, P. J. Maddon, R. A. Koup, J. P. Moore, et al. 1996. HIV-1 entry into by guest on September 27, 2021 similar expression patterns, we would predict that the majority of ϩ ϩ Ϫ CD4 cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667. Bonzo T cells will be compartmentalized to the CCR7 memory 15. Doranz, B. J., J. Rucker, Y. Yi, R. J. Smyth, M. Samson, S. C. Peiper, subset, suggesting a role for Bonzo in recruiting memory T cells to M. Parmentier, R. G. Collman, and R. W. Doms. 1996. A dual-tropic primary HIV-1 isolate that uses fusin and the ␤-chemokine receptors CKR-5, CKR-3, and sites of inflammation. CKR-2b as fusion cofactors. Cell 85:1149. In homozygous Bonzo knockout mice we have not yet detected 16. Choe, H., M. Farzan, Y. Sun, N. Sullivan, B. Rollins, P. D. Ponath, L. Wu, any functional defect. This may be due to redundancy in the che- C. R. Mackay, G. LaRosa, W. Newman, et al. 1996. The ␤-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135. mokine receptor system. However, it will be important to perform 17. Rucker, J., A. L. Edinger, M. Sharron, M. Samson, B. Lee, J. F. Berson, Y. Yi, more detailed analyses of migration patterns of T cells in response B. Margulies, R. G. Collman, B. J. Doranz, et al. 1997. Utilization of chemokine to a wide variety of pathogens and in particular to study T cell receptors, orphan receptors, and herpesvirus-encoded receptors by diverse human and simian immunodeficiency viruses. J. Virol. 71:8999. memory responses to the pathogens. Mutations in other chemokine 18. Unutmaz, D., V. N. KewalRamani, and D. R. Littman. 1998. G protein-coupled receptor family members, such as CCR5 and CCR2, resulted in receptors in HIV and SIV entry: new perspectives on lentivirus-host interactions phenotypes revealed only after studies in responses to pathogen or and on the utility of animal models. Semin. Immunol. 10:225. 19. Choe, H., K. A. Martin, M. Farzan, J. Sodroski, N. P. Gerard, and C. Gerard. in disease models (55–58). It will also be interesting to establish 1998. Structural interactions between chemokine receptors, gp120 Env and CD4. multiple knockouts between the Bonzo mutant mice and mutants Semin. Immunol. 10:249. for other chemokine receptors expressed in memory T cells to 20. Deng, H. K., D. Unutmaz, V. N. KewalRamani, and D. R. Littman. 1997. Ex- pression cloning of new receptors used by simian and human immunodeficiency identify potentially overlapping functions of these molecules. viruses. Nature 388:296. Bonzo is unique in that its sequence bears a noncanonic DRY 21. Loetscher, M., A. Amara, E. Oberlin, N. Brass, D. Legler, P. Loetscher, M. box motif that is thought to couple chemokine receptors to G pro- D’Apuzzo, E. Meese, D. Rousset, J. L. Virelizier, et al. 1997. TYMSTR, a putative chemokine receptor selectively expressed in activated T cells, exhibits teins (18, 20). It is currently not known whether Bonzo elicits HIV-1 coreceptor function. Curr. Biol. 7:652. signals that are similar to those of other chemokine receptors. It 22. Liao, F., G. Alkhatib, K. W. Peden, G. Sharma, E. A. Berger, and J. M. Farber. 1997. STRL33, a novel chemokine receptor-like protein, functions as a fusion will be critical to identify the natural ligand(s) of Bonzo to under- cofactor for both macrophage-tropic and T cell line-tropic HIV-1. J. Exp. Med. stand the receptor’s signaling function. In addition, mice in which 185:2015. the Bonzo gene has been replaced with EGFP may be of consid- 23. Alkhatib, G., F. Liao, E. A. Berger, J. M. Farber, and K. W. Peden. 1997. A new SIV coreceptor, STRL33. Nature 388:238. erable utility for tracking the migration patterns of memory T cell 24. Lucas, C., L. N. Bald, B. M. Fendly, M. Mora-Worms, I. S. Figari, E. J. Patzer, in responses to inflammatory stimuli. New insight into the expres- and M. A. Palladino. 1990. The autocrine production of transforming growth sion pattern and function of Bonzo may also help elucidate its role factor-␤1 during lymphocyte activation: a study with a monoclonal antibody- based ELISA. J. Immunol. 145:1415. during SIV/HIV infection. 25. Unutmaz, D., V. N. KewalRamani, S. Marmon, and D. R. Littman. 1999. Cyto- kine signals are sufficient for HIV-1 infection of resting human T lymphocytes. Acknowledgments J. Exp. Med. 189:1735. 26. Unutmaz, D., P. Pileri, and S. Abrignani. 1994. Antigen-independent activation We thank Drs. Chris Arendt and Monica Tsang for their critical reading of of naive and memory resting T cells by a cytokine combination. J. Exp. Med. the manuscript and helpful suggestions, Dr. Michael Black for providing 180:1159. 3292 REGULATION AND EXPRESSION PATTERN OF BONZO//STRL33

27. Unutmaz, D., V. N. KewalRamani, S. Marmon, and D. R. Littman. 1999. Cyto- 44. Oin, S., G. LaRosa, J. J. Campbell, H. Smith-Heath, N. Kassam, X. Shi, L. Zeng, kine signals are sufficient for HIV-1 infection of resting human T lymphocytes. E. C. Buthcher, and C. R. Mackay. 1996. Expression of monocyte chemoattrac- J. Exp. Med. 189:1735. tant protein-1 and interleukin-8 receptors on subsets of T cells: correlation with 28. Gu, H., Y. R. Zou, and K. Rajewsky. 1993. Independent control of immunoglob- transendothelial chemotactic potential. Eur. J. Immunol. 26:640. ulin switch recombination at individual switch regions evidenced through Cre-lox 45. Rabin, R. L., M. K. Park, F. Liao, R. Swofford, D. Stephany, and J. M. Farber. P mediated gene targeting. Cell 73:1155. 1999. Chemokine receptor responses on T cells are achieved through regulation 29. Beverley, P. C. 1992. Functional analysis of human T cell subsets defined by of both receptor expression and signaling. J. Immunol. 162:3840. CD45 isoform expression. Semin. Immunol. 4:35. 30. Bleul, C. C., L. Wu, J. A. Hoxie, T. A. Springer, and C. R. Mackay. 1997. The 46. Liao, F., R. L. Rabin, C. S. Smith, G. Sharma, T. B. Nutman, and J. M. Farber. 1999. CC-chemokine receptor 6 is expressed on diverse memory subsets of T HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on ␣ human T lymphocytes. Proc. Natl. Acad. Sci. USA 94:1925. cells and determines responsiveness to macrophage inflammatory protein 3 . 31. Liu, R., W. A. Paxton, S. Choe, D. Ceradini, S. R. Martin, R. Horuk, J. Immunol. 162:186. M. E. MacDonald, H. Stuhlmann, R. A. Koup, and N. R. Landau. 1996. Ho- 47. Huang, Y., W. A. Paxton, S. M. Wolinsky, A. U. Neumann, L. Zhang, T. He, mozygous defect in HIV-1 coreceptor accounts for resistance of some multiply- S. Kang, D. Ceradini, Z. Jin, K. Yazdanbakhsh, et al. 1996. The role of a mutant exposed individuals to HIV-1 infection. Cell 86:367. CCR5 allele in HIV-1 transmission and disease progression. Nat. Med. 2:1240. 32. Loetscher, P., M. Seitz, M. Baggiolini, and B. Moser. 1996. Interleukin-2 regu- 48. Dean, M., M. Carrington, C. Winkler, G. A. Huttley, M. W. Smith, R. Allikmets, lates CC chemokine receptor expression and chemotactic responsiveness in T J. J. Goedert, S. P. Buchbinder, E. Vittinghoff, E. Gomperts, et al. 1996. Genetic lymphocytes. J. Exp. Med. 184:569. restriction of HIV-1 infection and progression to AIDS by a deletion allele of the 33. Perera, L. P., C. K. Goldman, and T. A. Waldmann. 1999. IL-15 induces the CKR5 structural gene: Hemophilia Growth and Development Study, Multicenter expression of chemokines and their receptors in T lymphocytes. J. Immunol. AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City 162:2606. Cohort, ALIVE Study. Science 273:1856. 34. Jinquan, T., S. Quan, G. Feili, C. G. Larsen, and K. Thestrup-Pedersen. 1999. 49. Unutmaz, D., and D. R. Littman. 1997. Expression pattern of HIV-1 coreceptors Eotaxin activates T cells to chemotaxis and adhesion only if induced to express on T cells: implications for viral transmission and lymphocyte homing. Proc. CCR3 by IL-2 together with IL-4. J. Immunol. 162:4285. Natl. Acad. Sci. USA 94:1615. 35. Jourdan, P., C. Abbal, N. Nora, T. Hori, T. Uchiyama, J. P. Vendrell, J. Bousquet, N. Taylor, J. Pene, and H. Yssel. 1998. IL-4 induces functional cell-surface ex- 50. Baba, M., T. Imai, M. Nishimura, M. Kakizaki, S. Takagi, K. Hieshima, Downloaded from pression of CXCR4 on human T cells. J. Immunol. 160:4153. H. Nomiyama, and O. Yoshie. 1997. Identification of CCR6, the specific receptor 36. Wang, J., A. Harada, S. Matsushita, S. Matsumi, Y. Zhang, T. Shioda, Y. Nagai, for a novel lymphocyte-directed CC chemokine LARC. J. Biol. Chem. and K. Matsushima. 1998. IL-4 and a glucocorticoid up-regulate CXCR4 expres- 272:14893. sion on human CD4ϩ T lymphocytes and enhance HIV-1 replication. J. Leuko- 51. Loetscher, M., P. Loetscher, N. Brass, E. Meese, and B. Moser. 1998. Lympho- cyte Biol. 64:642. cyte-specific chemokine receptor CXCR3: regulation, chemokine binding and 37. Valentin, A., W. Lu, M. Rosati, R. Schneider, J. Albert, A. Karlsson, and gene localization. Eur. J. Immunol. 28:3696. G. N. Pavlakis. 1998. Dual effect of interleukin 4 on HIV-1 expression: impli- 52. Signoret, N., J. Oldridge, A. Pelchen-Matthews, P. J. Klasse, T. Tran, L. F. Brass,

cations for viral phenotypic switch and disease progression. Proc. Natl. Acad. Sci. M. M. Rosenkilde, T. W. Schwartz, W. Holmes, W. Dallas, et al. 1997. Phorbol http://www.jimmunol.org/ USA 95:8886. esters and SDF-1 induce rapid endocytosis and down modulation of the chemo- 38. Gosling, J., F. S. Monteclaro, R. E. Atchison, H. Arai, C. L. Tsou, kine receptor CXCR4. J. Cell Biol. 139:651. M. A. Goldsmith, and I. F. Charo. 1997. Molecular uncoupling of C-C chemokine receptor 5-induced chemotaxis and signal transduction from HIV-1 coreceptor 53. Signoret, N., M. M. Rosenkilde, P. J. Klasse, T. W. Schwartz, M. H. Malim, activity. Proc. Natl. Acad. Sci. USA 94:5061. J. A. Hoxie, and M. Marsh. 1998. Differential regulation of CXCR4 and CCR5 39. Solari, R., R. E. Offord, S. Remy, J. P. Aubry, T. N. Wells, E. Whitehorn, endocytosis. J. Cell Sci 111:2819. T. Oung, and A. E. Proudfoot. 1997. Receptor-mediated endocytosis of CC- 54. Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two chemokines. J. Biol. Chem. 272:9617. subsets of memory T lymphocytes with distinct homing potentials and effector 40. Amara, A., S. L. Gall, O. Schwartz, J. Salamero, M. Montes, P. Loetscher, functions. Nature 401:708. M. Baggiolini, J. L. Virelizier, and F. Arenzana-Seisdedos. 1997. HIV coreceptor 55. Kurihara, T., G. Warr, J. Loy, and R. Bravo. 1997. Defects in macrophage re- downregulation as antiviral principle: SDF-1␣-dependent internalization of the cruitment and host defense in mice lacking the CCR2 chemokine receptor. J. Exp.

chemokine receptor CXCR4 contributes to inhibition of HIV replication. J. Exp. Med. 186:1757. by guest on September 27, 2021 Med. 186:139. 56. Zhou, Y., T. Kurihara, R. P. Ryseck, Y. Yang, C. Ryan, J. Loy, G. Warr, and 41. Alkhatib, G., M. Locati, P. E. Kennedy, P. M. Murphy, and E. A. Berger. 1997. R. Bravo. 1998. Impaired macrophage function and enhanced T cell-dependent HIV-1 coreceptor activity of CCR5 and its inhibition by chemokines: indepen- immune response in mice lacking CCR5, the mouse homologue of the major dence from G protein signaling and importance of coreceptor downmodulation. HIV-1 coreceptor. J. Immunol. 160:4018. Virology 234:340. 42. Mack, M., B. Luckow, P. J. Nelson, J. Cihak, G. Simmons, P. R. Clapham, 57. Huffnagle, G. B., L. K. McNeil, R. A. McDonald, J. W. Murphy, G. B. Toews, N. Signoret, M. Marsh, M. Stangassinger, F. Borlat, et al. 1998. Aminooxypen- N. Maeda, and W. A. Kuziel. 1999. Cutting edge: Role of C-C chemokine re- tane-RANTES induces CCR5 internalization but inhibits recycling: a novel in- ceptor 5 in organ-speciﬁc and innate immunity to Cryptococcus neoformans. hibitory mechanism of HIV infectivity. J. Exp. Med. 187:1215. J. Immunol. 163:4642. 43. Sallusto, F., A. Lanzavecchia, and C. R. Mackay. 1998. Chemokines and che- 58. Traynor, T. R., W. A. Kuziel, G. B. Toews, and G. B. Huffnagle. 2000. CCR2 mokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol. expression determines T1 versus T2 polarization during pulmonary Cryptococcus Today 19:568. neoformans infection. J. Immunol. 164:2021.