CXCR4 and CCR5 mediate homing of primitive bone marrow–derived hematopoietic cells to the postnatal thymus

Experimental Hematology 34 (2006) 308–319

CXCR4 and CCR5 mediate homing of primitive bone marrow–derived hematopoietic cells to the postnatal thymus Paul Robertsona, Terry K. Meansb, Andrew D. Lusterb, and David T. Scaddena a

b

Center for Regenerative Medicine and Technology, Massachusetts General Hospital and Harvard Medical School, Boston Mass., USA; Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital and Harvard Medical School, Charlestown, Mass., USA (Received 28 June 2005; revised 10 November 2005; accepted 15 November 2005)

Factors governing the entry of cells into the postnatal thymus are poorly understood. We aimed to define molecular mechanisms mediating the homing of bone marrow cells to the thymus using a sublethally irradiated in vivo murine model. Entry of unfractionated and lineage-depleted bone marrow cells to the thymus, but not bone marrow, was a Gai-mediated phenomenon. Lineage-depleted cells that had homed to the thymus expressed abundant CXCR4 and CCR5 mRNA, alone of 17 chemokine receptors evaluated by QPCR. Thymichomed cells were distinct from cells that had homed to bone marrow in expression of CXCR4 and CCR5 by mRNA quantification and cell-surface expression of protein. Abrogation of CXCR4 and CCR5 function by genetic, antibody, or pharmacologic means impaired homing of lineage-depleted cells to the thymus, although not in a synergistic manner, implying interdependency of these receptors in the homing process. Competitive repopulation experiments demonstrated that inhibiting CXCR4-mediated homing adversely affected the double-negative cell pool at 2 weeks, suggesting that cells with prothymocytic activity may in part home via CXCR4. Overall, our data demonstrate differential homing mechanisms governing entry of unfractionated and lineage-depleted cells to irradiated bone marrow or thymus, with thymic homing of immature cells being pertussis-sensitive and mediated by the chemokine receptors CXCR4 and CCR5. Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc.

Since no self-renewing cells are resident in the thymus, continual recruitment of bone marrow–derived prothymocytes to the thymus is important for ongoing thymocyte production [1] and, therefore, maintaining immunologic homeostasis. Mechanisms governing cell localization to many organs including bone marrow (BM) [2] and lymph nodes [3] are beginning to be understood, but little is yet known about cell trafficking to the thymus. Several factors combine to make investigating thymic homing problematic. First, the thymus is not constantly receptive to the entry of progenitor cell, but permits cell entry only during transient, cyclical time periodsda process termed themic gating [4,5]. Gating is assumed to be achieved by transient expression of molecules known to be capable of inducing transendothelial migration in other organs, such as selectins, integrins, and chemokines, although the precise nature Offprint requests to: David T. Scadden, M.D., Center for Regenerative Medicine and Technology, 185 Cambridge Street, CP2N-4265A Massachusetts General Hospital and Harvard Medical School, Boston MA 02114; E-mail: [email protected]

of these molecules has yet to be defined [4,6]. Therefore, ensuring the entry of prothymocytes requires either careful timing of cell injection with thymic gating [4] or sublethal irradiation of recipient mice to allow synchronization of the thymic gate [5]. Second, the number of progenitor cells entering the thymus is small relative to other organs, limiting further manipulation or analysis of cells. Third, the thymus is a relatively inaccessible organ, making sequential sampling or imaging problematic in a live animal. Finally, the phenotype of the prothymocyte has not been definitively identified. In bone marrow, prothymocytic activity is found primarily in the lin2Sca-1Dc-kithiThy-12 compartment [7], supported by evidence that these early lymphoid progenitors (ELPs) are phenotypically and functionally similar to the earliest thymic progenitors (ETPs) [8]. This population can be further refined by expression of L-selectin [7]. A more mature cell, the Sca-12c-kit2B220D expressing common lymphoid progenitor-2 (CLP-2), also efficiently localizes to the thymus [9], but undergoes little proliferation once in the thymus and is less efficient at generating T cells, making it unlikely to represent the true prothymocyte.

0301-472X/06 $–see front matter. Copyright Ó 2006 International Society for Experimental Hematology. Published by Elsevier Inc. doi: 10.1016/j.exphem.2005.11.017

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Thymic immigrant cells at early time points have been shown to be c-kit2 [9,10], but more recently further subcategorization of CD252CD442 cells (DN1 cells) on the basis of c-kit and CD24 cells have been suggested that a small subset DN1 cells expressing c-kit represents possess the most robust prothymocytic activity [11]. Therefore, to address thymic homing in vivo requires circumvention of thymic gating, low thymic homing frequency, uncertainty of the phenotype of the prothymocyte, and relative inaccessibility of the thymus. Accordingly, most work attempting to define mechanisms of thymic homing has used in vitro models based on migration of cells into fetal thymic fragments. These studies have implicated the involvement of chemokines, a family of small proteins that induce directional movement of cells and that govern the migration of cells within [12–14] and from [15] the thymus, in cell trafficking to the thymus by the observation that migration of primitive cells into fetal thymic fragments in vitro depends on diffusible signals and occurs in a Gaidependent manner [16,17]. However, entry does not involve CCR9/CCL25 alone [16] but may involve both CCR9/ CCL25 and CCR7/CCL21 [17]. In vitro modeling has been supplemented by examination of thymi in various chemokine knockout mice. Mice deficient in CXCR4 [18], CCR9 [19], or CCR7 [20] have no overtly abnormal thymic phenotype, suggesting a minimal role of these molecules in thymic reconstitution. However, CXCR42/2 mice alone of the three are not viable into adulthood, and so while CXCR4 may not be important for fetal thymic reconstitution, it remains possible that CXCR4 plays a role in adult prothymocyte localization. Indeed, adoptive transfer of CXCR4-deficient cells, both CXCR42/2 FL [21] and SDF-intrakine transduced adult bone marrow cells [22] into adult wild-type mice resulted in reduced thymic cellularity, suggesting a role for CXCR4 in either the vascular delivery or proliferative capacity of progenitors. CXCR4 has subsequently been shown to be critical for thymocyte maturation, and possibly progenitor recruitment, in the adult thymus [13], contrasting with its limited role in fetal T lymphopoiesis [23,24]. Therefore there is mounting evidence that CXCR4 plays different roles in thymocytopoiesis during ontogeny, a phenomenon seen in other areas of hematopoiesis [25]. Given that the early fetal thymus is seeded by hematopoietic cells before it become vascularized, it would not be surprising that mechanisms of cell homing in this situation differ from those present in the fully vascularized adult thymus. Positive evidence in postnatal thymus is limited to a handful of molecules. b1 integrin is required for thymocytopoiesis in fetal and adult development [26]. L-selectin delineates a subset of Sca-1Dc-kitDThy-12lin2 cells with robust thymic reconstituting capacity [27], and the L-selectin ligand MECA-79 is found on thymic endothelium and is upregulated at times of importation of thymic progenitors [28], providing circumstantial evidence of an involvement

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in homing. CXCR4 has been suggested to play a role in immature thymocyte localization [13], but there has been no direct demonstration of either CXCR4 or L-selectin in thymic homing. Here, we use an in vivo homing model incorporating sublethal. While this model is imperfect, it provides an experimentally tractable system for cells arriving in the thymus under pathologic if not normal homeostatic conditions. The use of radiation allows synchronization of thymic gating in the recipient mice, providing us with a low but sufficient frequency of homing to permit comparisons of genetically and pharmacologically manipulated cells. In this way, we have identified molecular mechanisms of homing of unfractionated and phenotypically primitive bone marrow cells to the adult murine thymus, demonstrating a role for Gai-mediated pathways and, more specifically, the chemokine receptors CXCR4 and CCR5.

Materials and methods Mice C57BL/6, B6.SJL, RAG-22/2, B6129PF2, and CCR52/2 (B6; 129P2-Ccr5th1Kuz/J) mice were obtained from the Jackson Laboratories (Bar Harbor, ME, USA), and housed at the MGH CNY149 animal facility under specific pathogen-free conditions. Mice were irradiated using a 137Cesium source. Female mice, aged 4 to 10 weeks, were used in all experiments. Antibodies and inhibitors The following anti-murine monoclonal antibodies were used: biotinylated anti-CD3e (145-211C), CD4 (GX1.5), CD8b (53-6.7), CD11b (M1/70), CD19 (1D3), pan-NK (DX5), Gr-1 (RB6-8C5), Ter119 (Ly-76) and B220 (RA3-6B2), FITC-conjugated B220 (RA3-6B2), PE-conjugated CD45.1 (A20), FITC-conjugated CD45.2 (104), FITC-conjugated Sca-1 (E13-161.7), APC-conjugated c-kit (2B8), unconjugated and PE-conjugated anti-CXCR4 (2B11), biotinylated anti-CCR5 (C34-3448), and unconjugated IgG2b (all BD Pharmingen, San Jose, CA, USA). StreptavidinAPC and streptavidin PE-Cy7 were used as secondary antibodies (BD Pharmingen). Except where specified in the text, the lineage cocktail used was CD3, CD8, pan-NK, Gr-1, Mac-1, Ter119, and B220. Pertussis toxin (PTX) (Sigma-Aldrich, St. Louis, MO, USA) was used at a concentration of 1 mg/mL, and T140 at 10 mmol/mL. Homing assay Bone marrow cells were harvested from donor mice by flushing tibial and femoral bone shafts with RPMI containing 10% fetal calf serum, and lysed with ACK. In certain experiments, lineage depletion was performed as described below. Cells were resuspended in warmed phosphate-buffered saline (PBS) and incubated with 2 mmol/mL CFDA-SE (Molecular Probes, Eugene, OR, USA) for 15 minutes at 37 C. Cells were washed and incubated in medium for a further 30 minutes. In certain experiments cells were further incubated with pertussis toxin (1 mmol/mL), T140 (10 mmol/mL), an anti-CXCR4 antibody (2B11, 10 mg/mL), or an isotype control antibody (IgG2b, 10 mg/mL) for 1 hour at 37 C. Cells were counted using trypan blue staining and

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suspended at the desired concentration in serum-free RPMI. Cells were filtered through a 20-mm mesh and 0.5 to 1.0 3 106 cells injected into the lateral tail vein of age-matched recipient mice, 4 to 6 hours after irradiation. Lineage depletion Following lysis with AKC, whole BM cells were incubated with a saturating concentration of lineage-specific antibodies (CD3, CD8, pan-NK, Gr-1, Mac-1, Ter119, and B220) for 15 minutes at 4 C. Cells were washed and run through LD depletion columns (Miltenyi Biotech, Auburn, CA, USA) according to the manufacturer’s instructions. Analysis of homing cells Mice were euthanized 20 hours following adoptive transfer and single cell suspensions were made from bone marrow, thymus, and, in some experiments, spleen. Care was taken to strip the thymus of attached connective tissue. Red cells in splenic and bone marrow cell suspensions were lysed with ACK. Cell counts were performed following staining with trypan blue, and fixed with 2% paraformaldehyde. Flow cytometric analysis was performed within 24 hours using a FACScalibur (BD Biosciences) flow cytometer. Live cells were gated based on forward-scatter (FSC) and side-scatter (SSC) parameters. Files of at least 2.5 3 105 events were collected for BM and spleen, and 1 3 106 for thymus. Quantitative PCR One 3 106 lineage-depleted cells were labeled with CFDA-SE and injected into sublethally irradiated mice. At 20 hours bone marrow and thymus were harvested, and single cell suspensions made. CFSED cells were sorted using a FACSVantage (BD Biosciences) cell sorter. RNA was extracted from sorted cells using the RNeasy kit according to manufacturer’s instructions (Qiagen, Valencia, CA, USA) and quantitative PCR (QPCR) performed as previously described [29]. Immunophenotyping of homed cells One femur-equivalent of whole bone marrow from a CD45.1 (B6.SJL) mouse was injected into an age-matched sublethally irradiated CD45.2 (C57BL/6) mouse. Thymus and bone marrow were harvested from recipient mice at 20 hours and single cell suspensions made by gentle mechanical disruption. BM cells were lysed with ACK. Cells were counted, resuspended in PBS, and treated with FcR block (BD Pharmingen) for 15 minutes at 4 C. Cells were then stained with CD45.1-PE, CD45.2-FITC, CXCR4-PE, lineage (CD3, CD8, CD19, Gr-1, Mac-1, NK-1, Ter119) biotin, CCR5-biotin, B220-FITC, Sca-1-FITC, or c-kit-APC. Streptavidin-APC was used as a secondary antibody for biotinylated antibodies. Chemotaxis assay Following ACK lysis 2 to 5 3 106 whole bone marrow cells were incubated for 1 hour at 37 C with either no treatment, pertussis toxin, T140, or SDF-1a. Treated cells were washed twice in PBS. Twenty-four-well Transwell plates (Corning, Acton, MA, USA) with a 5-mm membrane pore size were used. Next. 0.5 to 1 3 105 cells in 150-mL medium were pipetted into the upper wells. Five hundred mL RPMI alone or RPMI with 100 ng/mL SDF-1a (R&D Systems, Minneapolis, MN, USA) was added to the lower wells. Plates were incubated at 37 C for 3 hours. Cells

in the lower well were counted and percentage migration calculated based on the number of input cells. Competitive thymic repopulation assay Unfractionated bone marrow cells were obtained from C57BL6 (CD45.2) and B6.SJL (CD45.1). CD45.2 cells were incubated with either IgG2b (10 mg/mL) or 2B11 (10 mg/mL) for 1 hour at 37 C. All CD45.1 cells were incubated with IgG2b antibody (10 mg/mL). Cells were mixed at a 1:1 ratio and 0.5 3 106 total cells injected via lateral tail veins into RAG-22/2 mice (CD45.2), lethally irradiated (9.5 Gy) 6 hours prior to adoptive transfer. An aliquot of injectate was taken to confirm the expected ratio of cells by flow cytometry. Mice were euthanized at 2 or 4 weeks. Thymi, bone marrow, and spleen were harvested and analyzed for expression of CD45.1 and CD45.2 by flow cytometry. Thymi were also analyzed for CD4 and CD8 expression. Data analysis Data were analyzed using Microsoft Excel (Microsoft, Redmond, WA, USA), and reported as mean 6 SEM throughout. Significance calculations were performed using the two-tailed Student’s t-test, a 5 0.05.

Results Immature cells entering the thymus are predominantly large Sca-1Dc-kit2B2202 cells Unfractionated bone marrow cells and lineage-depleted cells from 4- to 8-week-old C57BL/6 mice were isolated, stained with 2 mmol/mL CFDA-SE, and injected into agematched congenic mice irradiated with 3.5 Gy 4 to 6 hours prior to injection. Bone marrow and thymus were harvested at 20 hours and analysis by flow cytometry showed a clear subpopulation of CFSED cells (Fig. 1A), not present in control injections performed with unlabeled cells (data not shown). The fluorescence of the CFSED cells at 20 hours was unchanged compared with cells analyzed prior to injection, indicating that no cell division was occurring that could cause a falsely high frequency of homed cells. Of note, lineage-depleted cells homing to the thymus had a characteristic scatter plot representing large cells when compared to the more heterogeneous scatter plots seen with injection of unfractionated bone marrow cells (Fig. 1B). Prothymocytes have previously been noted to be large cells [30–32], and these homing cells have equivalent scatter plots to double-negative (DN) 1-2 cells in nonirradiated wild-type thymus (data not shown). To phenotype the homed cells, one femur equivalent of unfractionated bone marrow cells from CD45.1 mice was injected into sublethally irradiated CD45.2 mice. Cells were harvested from bone marrow and thymus of recipient mice at 20 hours and stained with CD45.1-PE, a biotinylated lineage cocktail substituting CD19 for B220, Sca-1-FITC, B220-FITC, and c-kit-APC. Gates for B220, c-kit, and Sca-1 were set using IgG isotype staining of homed CD45.2 cells. Lineage-negative cells represented 18% of bone marrow homing cells and 23% of thymic homing cells

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Figure 1. Phenotypic characterization of lineage-depleted cells homing to thymus. (A): Flow cytometric analysis of harvested thymus 20 hours after adoptive transfer of CFDA-SE-labeled cells shows a clearly identifiable population of CFDA-SED cells (Gate R2). (B) shows scatter plots of these CFDA-SED cells (Gate R2 in A) demonstrating that lineage-depleted input cells homing to the thymus are larger and less heterogeneous in size than unfractionated bone marrow cells. In (C) homed cells gated to exclude cells expressing lineage markers were analyzed for expression of Sca-1, c-kit, and B220. Quadrant lines were based on staining of isotype control antibodies Data shown are representative of 3 mice, 2 independent experiments.

and, of these lineage-negative cells homing to the thymus, 69% 6 1.5% were Sca-1Dc-kit2, and 20% 6 3.0% were B220Dc-kit2, with 2% 6 0.5% Sca-1Dc-kitD (Fig. 1C). This was contrasted by bone marrow homing where a greater proportion of cells expressed B220 (68% 6 14%) and c-kit (5%) but fewer cells were Sca-1D (18.7% 6 2.6%). This phenotype of thymic-homed cells is in keeping with previous findings [10] but inconsistent with the ELP phenotype defined as Sca-1Dc-kitD [8], and the finding of prothymocytic activity exclusively in c-kitD primitive bone marrow cells [7]. It is possible that c-kit is lost in the homing process before being upregulated following thymic entry, or that the small number of c-kit-expressing cells do represent the true prothymocyte which require several days to proliferate to detectable levels.

Unfractionated and lineage-depleted cell homing to the thymus is Gai dependent Given the ability of PTX to inhibit cell traffic into fetal thymic fragments in vitro, and within and from the thymus in vivo, we postulated that PTX might also inhibit entry to the adult thymus in vivo. Pertussis toxin is a specific inhibitor of the Gai subunit of G-protein-coupled receptors that include chemokine receptors. Cells were pretreated with 1 mg/mL pertussis toxin for 1 hour prior to injection into the recipient mouse. Cells were washed thoroughly to remove traces of PTX. PTX treatment had no significant effect on homing of whole bone marrow cells to the bone marrow and increased homing to the spleen by 2.7-fold at 4 hours (p 5 0.009) and 3.0 at 20 hours (p 5 0.0005); however, PTX treatment profoundly reduced homing to

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the thymus for unfractionated bone marrow cells at both 4 (84.5 6 12.7 to 5.0 6 2.2, p 5 0.00004) and 20 hours (202 6 26 to 30 6 11, p 5 0.0015) (Fig. 2A). PTX treatment also reduced lineage-depleted bone marrow cells homing to the thymus from 495 6 87 cells/million thymocytes to 20 6 6 cells/million, a 96% reduction (p 5 0.03) (Fig. 2B, left panel), but PTX-treated cells homed normally to both bone marrow and spleen (Fig. 2B, right panel). Total cell number paralleled frequency, with 1828 6 267 untreated cells homing and only 140 6 57 PTX-treated cells, a reduction of 92% (p 5 0.02, data not shown). Thus we conclude that the lineagedepleted bone marrow compartment is enriched for cells capable of thymic homing, and that entry of both mature and primitive cells to the thymus is a Gai-mediated event. CXCR4 and CCR5 mRNA expression in thymic-homed lineage-depleted cells Many receptors signal through Gai-mediated pathways, but we hypothesized that chemokine receptors were, at least in part, responsible for thymic homing. Given that thymic homing, but not bone marrow homing, was PTX dependent, we reasoned that differential chemokine receptor expression might be found between bone marrow–homed and thymic-homed cells. We therefore performed a screen of 17 chemokine receptors, comparing lineage-depleted cells having homed to the thymus with those having homed to the bone marrow. One 3 106 lineage-depleted cells were

isolated from C57BL/6 mice, CFDA-SE labeled as before, and injected into sublethally irradiated congenic agematched recipient mice. At 20 hours, bone marrow and thymus were harvested and 1000 CFDA-SED cells were sorted from both thymus and bone marrow. RNA was isolated and quantitative PCR was performed for 17 chemokine receptors, b2-microglobulin, and GAPDH. Each set of homed cells was also compared to the input lineagedepleted population. Of the 17 chemokine receptors tested, mRNA from each was present in small amounts in the input cells and in the bone marrow–homed cells, but none was predominant. In contrast, CXCR4 and CCR5 mRNA alone of the 17 chemokine receptors tested were abundantly expressed in thymic homing cells, with mean 3.8-fold expression over controls for CCR5 and mean 5.0-fold increase for CXCR4 (Fig. 3). Similar results were seen when b2 microglobulin rather than GAPDH was used as the housekeeping gene control, or when data was expressed as number of copies per cell. Cell-surface expression of CXCR4 and CCR5 on unfractionated thymic-homed cells Having demonstrated CXCR4 and CCR5 expression at an mRNA level, we next sought to establish whether this correlated to surface expression of these chemokine receptors. A CD45-disparate adoptive transfer model was again used. Using lineage-depleted input cells provided too few homed cells to perform reliable immunophenotyping, and so

Figure 2. Pertussis toxin inhibits homing of bone marrow cells to the thymus but not to bone marrow or spleen. The frequency of cell homing to thymus, bone marrow, and spleen following treatment with PTX (black bars) or mock treatment (white bars) is shown. Top panels (A) show homing of unfractionated bone marrow cells. Lower panels (B) show homing of lineage-depleted bone marrow cells. Data are shown as mean 6 SEM from 7 mice, 4 independent experiments (A) and 4 mice, 2 independent experiments (B).

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Figure 3. Lineage-depleted cells homing to thymus, but not bone marrow, express abundant CXCR4 and CCR5 mRNA. CFDA-SE-labeled, lineagedepleted cells were intravenously injected into sublethally irradiated mice. 1000 CFSED cells were sorted from harvested BM and thymus at 20 hours and RNA was extracted. QPCR for 17 chemokine receptors, b2m, and GAPDH was performed. Data shown are representative from 2 independent experiments, and show quantity of each chemokine receptor relative to GAPDH with black bars showing mRNA in thymic homed cells, and white bars showing mRNA in BM-homed cells.

unfractionated bone marrow cells from both femora and tibias from single CD45.2 donor was intravenously injected into a sublethally irradiated CD45.1 mouse. Bone marrow and thymus harvested at 20 hours were analyzed by flow cytometry. CD45.2D cells falling within a live cell gate (based on FSC and SSC properties) were analyzed for their expression of CCR5 and CXCR4. Gates for CCR5 and CXCR4 positivity were set based on background fluorescence of IgG isotype controls. A greater mean percentage of cells homing to the thymus were CXCR4D (78% 6 7%) compared with those in the bone marrow (Fig. 4) (15% 6 5%). The same was true for CCR5D cells (37% 6 13% vs 5 6 1%) (Fig. 4). CXCR4 and CCR5 mediate lineage-depleted cell homing to the thymus Having demonstrated that thymic homing, but not bone marrow homing, was Gai mediated, and that thymichoming cells, but not bone marrow–homing cells, express more CXCR4 and CCR5 as cell surface protein and mRNA, we assessed the role of these chemokine receptors in thymic homing. CXCR42/2 mice die in utero and, thus, adult bone marrow cells are not available [23]. Therefore, to abrogate CXCR4 function we used the anti-CXCR4 antibody, 2B11, which binds to both human and murine forms of CXCR4, and is effective at inhibiting chemotaxis of murine lineage-depleted bone marrow cells to 100 ng/mL SDF-1 in vitro (Fig. 5). We also used T140, a previously defined and highly specific pharmacologic inhibitor of CXCR4 [33,34] similarly effective in vitro to using a blocking antibody to CXCR4 (Fig. 5). CCR52/2 mice are viable into adulthood and possess only mild phenotypic abnormalities [35], and thus the capacity of CCR52/2 bone marrow cell homing could be tested directly. Unfractionated CCR52/2 bone marrow cells showed no significant impairment of homing to either bone

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marrow or thymus (Fig. 6). By flow cytometry, CXCR4 expression on CCR52/2 bone marrow cells is equivalent to wild-type mice and CCR52/2 mice do not possess fewer immature hematopoietic cells (data not shown). Lineagedepleted bone marrow cells from CCR52/2 mice or agematched wild-type mice were treated with either 2B11 or an isotype control antibody for 1 hour at 37 C, and injected into sublethally irradiated BL6 mice. Thymus and bone marrow were harvested at 20 hours and frequency of homing assessed by flow cytometry. Thymic homing was reduced in frequency from 244 6 14 cells per million thymocytes in control antibody-treated cells to 100 6 10 in 2B11-treated cells, a 59% reduction (p 5 0.00004), to 147 6 16 in CCR52/2 cells, a 40% reduction (p 5 0.002), and to 81 6 13 in CCR52/2 cells treated with 2B11, a 67% reduction (p 5 0.00003) (Fig. 7A). Total cell number paralleled frequency: 1540 6 118 control antibody–treated wild-type cells homed to the thymus at 20 hours, compared to 507 6 72 2B11treated wild-type cells, a reduction of 67% (p 5 0.0002), 623 6 176 control antibody–treated CCR52/2 cells, a reduction of 60% (p 5 0.003), and 406 6 102 2B11-treated CCR52/2 cells, a reduction of 74% (p 5 0.0001). There was no significant synergistic or even additive effect when CCR52/2 cells were treated with 2B11 (p 5 0.30), suggesting that either signaling via both CXCR4 and CCR5 is necessary for optimal homing or that inhibition of one ligand-receptor interaction adversely affects signaling through the other. The absence of a synergistic effect also argues against the possibility that two separate populations of cells using either CCR5 or CXCR4 for entry are present among the homed cells. Homing of lineage-depleted cells to bone marrow was CXCR4 dependent but CCR5 independent (Fig. 7B). Frequency of homing of control antibody–treated cells to bone marrow was 770 6 72 per million, 679 6 27 with CCR52/2 cells (p 5 0.30). Treatment of cells with 2B11 resulted in homing frequencies of 408 6 32 per million in wild-type cells (p 5 0.005) and 450 6 37 in CCR52/2 cells (p 5 0.001), reductions of 47% and 34% respectively. To validate that the effect of 2B11 was due to blocking and not nonspecific effects of the antibody on CXCR4expressing cells, we treated lineage-depleted cells with T140. Pretreatment of cells with T140 resulted in a reduction of thymic homing similar to 2B11-treated cells, suggesting that the abrogation was CXCR4 specific, not simply a nonspecific effect of antibody binding (Fig. 8). Unfractionated Abrogation of CXCR4-mediated homing affects short-term but not long-term thymic reconstitution Although the homing of lineage-depleted cells was CXCR4 dependent, we cannot assume that the true prothymocyte is similarly CXCR4 dependent given the heterogeneous nature of lineage-depleted cells entering the thymus [11]. To address this we employed a 4-week competitive

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Figure 4. A greater frequency of thymic-homed cells express CXCR4 and CCR5 than bone marrow–homed cells. Unfractionated bone marrow cells from BL6 mice (CD45.2) were injected into sublethally irradiated B6.SJL mice (CD45.1). Bone marrow and thymus were harvested at 20 hours and cells stained for CCR5, CXCR4-PE, and CD45.2 FITC. The figure shows the frequency of cell-surface expression of CCR5 and CXCR4 in cells having homed to thymus vs bone marrow. Data shown are means 6 SEM from 3 mice, 2 independent experiments.

reconstitution assay of CD45 disparate cells into lethally irradiated RAG-22/2 mice (CD45.2), which possess a developmental block preventing progression beyond the DN stage [36], and therefore any DP or SP cells present in reconstituted thymi must be donor derived. Ongoing absence of CXCR4 or CCR5 function, whether by pharmacological

Figure 5. T140 and 2B11 inhibit migration of unfractionated BM cells to SDF-1a in vitro. Chemotaxis assays were performed using unfractionated bone marrow cells treated with T140 (10 mmol/mL), 2B11 (10 mg/mL), IgG2b (10 mg/mL), or mock treated with PBS and placed in upper wells of a 5-mm Transwell plate. The number of cells migrating to medium alone or medium plus SDF-1a (100 ng/mL) at 3 hours was counted. Percentage migration was calculated by dividing the number of migrating cells by the number of input cells per well was calculated. Data are shown as means 6 SEM, 3 independent experiments performed in triplicate.

inhibitor or genetic modification of cells, may impair intrathymic development. Thus we would be unable to distinguish between reduced homing to the thymus or reduced intrathymic development as the cause for any delay in thymic reconstitution. Therefore we incorporated a competitive aspect to the reconstitution assay to allow us to evaluate the specific effect of homing on thymic reconstitution. We evaluated thymic, bone marrow, and splenic reconstitution of CD45.1 and CD45.2 cells injected in a 1:1 ratio into lethally irradiated mice. In the control arm both CD45.1 cells and CD45.2 cells were treated with an isotype control antibody, anticipating that the ratio of cells in the injectate would be mirrored by the ratio of cells in the reconstituted organs. In the test arm, CD45.2 cells were treated with anti-CXCR4 antibody and also injected in a 1:1 ratio with isotype control antibody–treated CD45.1 cells. If CXCR4 mediates homing of prothymocytes, CD45.1 control antibody–treated cells should out-compete CD45.2 cells treated with an anti-CXCR4 antibody for the limited prothymocytic niches within the thymus. Thus, if our hypothesis is valid, frequency of anti-CXCR4 antibody-treated CD45.2 DN and DP cells should be less than seen in isotype control–treated CD45.2 cells, while showing equal reconstitution of bone marrow and spleen. However, whether any isotype disparity would either persist beyond a brief interval in the DN population or be sustained as DN cells mature to DP cells could not be predicted given the potential for subsequent cell pool size modulation. We tested CXCR4 only as we had no validated means of transiently inhibiting CCR5 function in vivo. Whole bone marrow was obtained from BL6 (CD45.2) and SJL.B6 (CD45.1) mice. All CD45.1 cells were treated with an isotype control antibody at 10 mg/mL. A control group of CD45.2 cells was also treated with an isotype control antibody, and the test group with the anti-CXCR4 antibody, 2B11 (both at 10 mg/mL). Cells were mixed at a 1:1 ratio and injected into lethally irradiated mice and the thymi, bone marrow, and spleen of recipient mice were analyzed at 2 or 4 weeks. Given thymocyte development is crucially dependent on CXCR4 [5], we administered the anti-CXCR4 antibody only to cells prior to injection and did not continue administration beyond pretreatment. At 4 weeks we saw no significant difference in total cellularity of any of the organs examined, or the frequency of CD45 contribution in thymus, BM, or spleen. At 2 weeks no significant difference in cellularity was seen in either the isotype-treated or the 2B11-treated groups in any of the organs assessed, and the CD45 contribution in spleen, bone marrow, total thymocytes, or DP thymocytes approximated the 1:1 ratio of the injectate (data not shown). However, unexpectedly, we saw that in our control arm CD45.2 cells were over-represented among the DN population in the control mice (98.8% CD45.2), suggesting a competitive advantage of CD45.2 over CD45.1 in thymic engraftment

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Figure 6. Unfractionated CCR52/2 cells home normally to bone marrow, spleen, and thymus. Unfractionated bone marrow cells from wild type or CCR52/2 mice were CFDA-SE labeled and injected into sublethally irradiated mice. Frequency of thymic homing (left panel) and bone marrow and splenic homing (right panel) at 20 hours are shown. Data shown are means 6 SEM, n 5 4 from 2 independent experiments.

(Fig. 9). This difference was not sustained at 4 weeks. It is unclear whether the restoration of the expected ratio by 4 weeks was due to importation of a fresh cohort of host BM-derived cells, loss of CD45.2 DN cells by differentiation, or selective expansion of CD45.1 cells. This finding cannot be explained simply as persistence of host RAG-22/2 cells, as significant differences were seen in the frequency of CD45.2 cells in the anti-CXCR4-treated group, and the total number of DN cells exceeded that seen in thymi of nonreconstituted RAG-22/2 mice. Relative frequency of the different isoforms in bone marrow and spleen of the same mice approximated the 1:1 ratio of the injectate, eliminating the possibility that the frequency of CD45.2 cells was greater in the injectate. Given this competitive advantage of CD45.2 cells, the finding that the percentage of anti-CXCR4 antibody–treated CD45.2 DN cells is reduced compared with control CD45.2 cells (45.5% vs 98.8%) indicates that CXCR4 inhibition affected early events in thymic reconstitution, an effect

that was lost by 4 weeks. These results suggest that inhibiting CXCR4-mediated homing reduces the size of the DN pool at 2 weeks, an effect that is consistent with reduced thymocyte progenitors arriving in the thymic microenvironment. However, the DN pool will include other immature cells known to enter the thymus besides prothymocytes, including Sca-1Dc-kit2, Sca-12B220D cells whose role is of uncertain significance. Discussion Determining the factors mediating homing to the postnatal thymus is best achieved in an in vivo model, which requires circumvention of thymic gating, low thymic homing frequency, uncertainty of the phenotype of the prothymocyte, and relative inaccessibility of the thymus. We have chosen to use an in vivo homing model that, although imperfect, we believe sufficiently overcomes the above difficulties to permit useful information. Our model incorporates sublethal irradiation to allow synchronization of thymic gating,

Figure 7. Differential chemokine receptor involvement in thymic and bone marrow homing. Lineage-depleted cell homing to the thymus is dependent on CXCR4 and CCR5. Lineage-depleted CFSE-labeled bone marrow cells from either CCR52/2 mice (KO) or wild-type controls (WT) were injected into a sublethally irradiated recipient mouse. Prior to injection cells were treated with either an anti-CXCR4 antibody (2B11) or isotype control (IgG). The frequency of cell homing to thymus (A, left panel) and bone marrow (B, left panel) of recipient mice at 20 hours is shown. The right-hand panels show total number of cells homing to thymus (A) and bone marrow (B). Data is shown as means 6 SEM, n 5 6 from 3 independent experiments.

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Figure 8. T140, a small molecule inverse agonist of CXCR4, inhibits cell homing to the thymus. Homing assays were performed as previously described. Prior to injection, CFDA-SE labeled whole bone marrow cells were incubated with PBS, pertussis toxin (1 mmol/mL), or T140 (1 mmol/mL), labeled as untreated, PTX, and T140 respectively. Frequency of 4-hour homing to thymus (left panel) and BM and spleen (right panel) is shown. Data shown are the means 6 SEM, n 5 7 from 4 independent experiments.

without which it is difficult to be certain of thymic receptivity to progenitor cells. Sublethal irradiation has previously been shown not to influence the number or function of prothymocytic niches [5]. Although radiation induces increased permeability of local vessels, we observe different homing mechanisms mediating homing to thymus and bone marrow, suggesting that nonspecific effects of radiation do not account for the thymic homing mechanisms we describe. We show that thymic homing can be reliably tracked using CFSE labeling and CD45 disparate cells. To counter the uncertainty regarding phenotypic markers of the true prothymocyte, we have chosen to use the heterogeneous lineage-depleted cell group in order to be certain we included cells with prothymocytic activity that may be excluded by a positive selection strategy. We recognize that cells entering the thymus will be diverse, with only a minority being true prothymocytes, and accept that our findings cannot definitively provide evidence of prothymocytic homing mechanisms. We suggest that mechanisms outlined in this work could be extrapolated to prothymocytic homing, and provide a starting point for further studies in this area. We show that lineage-depleted cells are enriched for thymic homing capacity and that homed cells are predominantly large, Sca-1Dc-kit2 in keeping with other groups. We note two smaller populations; around 20% are B220D consistent with the CLP-2 [9], and 2% Sca-1Dc-kitD consistent with what others have defined as the true prothymocyte [11]. We show that homing of unfractionated and lineage-depleted bone marrow cells to the thymus involves Gai-mediated mechanisms by abrogating thymic homing with PTX pretreatment. Care was taken to strip connective tissue containing lymph nodes from thymi before processing to exclude PTX-mediated block of CCR7-driven lymphocyte homing to lymph nodes as an explanation for this finding. Further, lineage-depleted cells, containing few lymphocytes, displayed similar PTX sensitivity.

We reasoned that PTX sensitivity of homing was evidence of a chemokine-mediated process and chemokines signal predominantly through Gai-coupled pathways. Screening recently homed cells for a variety of chemokine receptors showed that those entering the thymus expressed abundant CXCR4 and CCR5 mRNA, whereas those recently entered to bone marrow did not. We chose to screen mRNA initially as absence of cell-surface chemokine receptor expression on recently homed cells would not necessarily rule out a role for chemokine receptors in homing, given that cells may rapidly undergo phenotypic change

Figure 9. Inhibition of CXCR4-mediated homing impairs early thymic reconstitution. Unfractionated bone marrow cells from C57BL6 (CD45.2) and B6/SJL (CD45.1) mice were obtained. All CD45.1 cells were incubated with an isotype control antibody. CD45.2 cells were incubated with the anti-CXCR4 antibody, 2B11, or an isotype control antibody, all at 10 mg/mL. CD45.1 and CD45.2 cells were mixed at a 1:1 ratio and 5 3 105 total cells injected into lethally irradiated RAG-22/2 mice. At 2 weeks (shown) or 4 weeks (not shown), thymus, bone marrow, and spleen were harvested and analyzed for percentage contribution of CD45.2 cells by flow cytometry. Only cells engrafting thymus demonstrated a difference and are shown. Data shown are means 6 SEM, n 5 6, 2 independent experiments.

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during transendothelial migration. Even if cell-surface receptors were downregulated, mRNA would remain for longer time periods. Further evidence for CXCR4 and CCR5 involvement in thymic homing of lineage-depleted cells was provided by showing enrichment of CXCR4 and CCR5 cell-surface protein in thymic-homed cells compared with bone marrow–homed cells. Loss-of-function studies demonstrated that both CXCR4 and CCR5 mediated the homing process. This is contrasted with homing to bone marrow where only CXCR4 was involved, but in a Gai-independent manner, a finding similar to human CD34 cell homing to NOD-SCID bone marrow [37]. The findings using an anti-CXCR4 antibody were duplicated using a highly specific pharmacologic inhibitor of CXCR4, suggesting that nonspecific effects of bound antibody was not an explanation for the effect of the 2B11 antibody. In addition, CCR52/2 cells did not appear to have fewer primitive bone marrow cells or altered CXCR4 expression, suggesting that the loss of homing seen in the CCR52/2 mice was solely due to absence of CCR5. It should also be noted that even with dual abrogation of CXCR4 and CCR5, a greater proportion of cells were still able to home to the thymus compared with GaI inhibition (40% vs 5%), and therefore there may remain Gaidependent receptors that contribute to thymic homing over and above those we have described, although we cannot exclude the possibility that our in vivo methods of chemokine receptor blockade were not maximally effective. The lack of synergism when both CCR5 and CXCR4 function were inhibited was unexpected and suggests that signaling from both receptors may be necessary for optimum homing to occur, and argues against the existence of two separate primitive populations of cells, one CXCR4 dependent and one CCR5 dependent, entering the thymus. That inhibition of one receptor could have an effect on the other is also possible, but we have no data to directly address that issue. Others have indicated that chemokine receptor heterodimerization may be a means of generating increased sensitivity to the relatively low amounts of chemokine fixed to apical endothelial surfaces and thereby induce a cooperative effect among receptors [38]. We speculate that heterodimerization between CCR5 and CXCR4 would be one explanation of our finding. Both molecules can co-localize in lymphocytes [39,40] and are capable of forming heterodimers with CCR2 [38]. In addition, CCR5 activation has been shown to modulate CXCR4 function [41]; however, no published evidence exists of CXCR4 and CCR5 forming heterodimers. In our final experiment we attempted to tease out the role of CXCR4-mediated homing in thymic reconstitution. At 2 weeks, blocking CXCR4-mediated homing causes a competitive disadvantage within the DN population, but this disadvantage is lost by 4 weeks. Further DP cell reconstitution is unaffected by CXCR4 blockade, suggesting that prothymocytes may enter by means other than CXCR4. This early difference can only be explained by differences in

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homing of cells, as no ongoing CXCR4 inhibition occurred in the experiment, excluding inhibition of CXCR4-induced intra-thymic proliferation as an explanation for the observed difference. However, we note that DP cell reconstitution is unaffected by CXCR4 blockade, suggesting that prothymocytes may enter by means other than CXCR4. Our findings of CXCR4 and CCR5 relevance in thymic homing require reconciliation with the absence of a clear thymic phenotype in mice that are deficient in either molecule or in humans with the CCR5D32 polymorphism [18,23,42]. First, the absence of the receptors in development may be overcome by compensatory use of other molecules that provide a functional redundancy. Second, a relative paucity of cells entering the thymus may be overcome by a compensatory expansion of that population to provide essentially normal homeostasis of progenitor pools. Third, it is possible that the initial investment of the thymic bud is accomplished by mechanisms that are independent of the mechanisms we describe in the adult and may be due to tissue migration distinct from the vascular transmigration we tested in the adult. Fourth, as discussed above, CXCR4 and CCR5 may only mediate the homing of the Sca-1Dc-kit2 cell population and not the true prothymocyte. Nevertheless, that CXCR4 and CCR5 play a role in cell homing to the adult thymus may have several implications. First, CXCR4 and CCR5 inhibitors are currently under development [43] may have a previously unanticipated effect on thymic reconstitution. Second, we note with interest that the two receptors highlighted by this study are HIV coreceptors [44–46]. Although they may not be similarly relevant to human thymic, this finding would provide a means by which the human immunodeficiency virus (HIV) could gain access to the immunologically privileged site of the thymus. HIV infects thymocytes early in infection and causes progressive loss of thymic function [47]. Further, they raise the possibility of HIV-1 having a further effect on the infected host’s ability to repair defects in T cell numbers by altering the capacity of bone marrow– derived precursors to home and engraft in the thymus. Third, if entry of mature cells to the thymus is involved in the pathophysiology of autoimmunity, manipulating CXCR4 may be a therapeutic means of restoring normal immune function. Finally, whether enhancing transplanted cells sensitivity to the chemokine signals reported here can affect thymic homing and thereby T-cell reconstitution in settings such as HIV disease or bone marrow transplantation remain to be explored. The results presented here may help guide such future studies.

Acknowledgments The authors thank David Dombkowski and Mike Waring for cell sorting expertise, Dr. Gregor B. Adams and Dr. Kenneth S. Cohen for helpful discussion, and Karissa Adams, Meagan Kilbride, and Chris Pasker for administrative assistance.

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