Human Lymphoid and Myeloid Cell Development in NOD/LtSz-scid IL2Rγ^null Mice Engrafted with Mobilized Human Hemopoietic Stem Cells ¹²

Hemopoietic stem cells (HSC) ⁴ for clinical transplantation can be obtained from bone marrow (BM), cytokine-mobilized peripheral blood, or umbilical cord blood (UMB; 1, 2, 3, 4). Myeloablative therapy followed by allogeneic HSC transplantation is curative for many hematological malignancies, genetic disorders, and autoimmune diseases (5, 6, 7). However, HSC transplantation has not realized its full potential due to the relatively low numbers of suitable donors, the technical difficulties associated with HSC transplantation, and the ethical limitations associated with in vivo studies in humans. Because of this latter constraint, many of the quantitative studies on human HSC have been performed using in vitro colony assays (2). However, investigating the in vivo engraftment and differentiation capability of human HSC remains essential for understanding the true potential of primitive human HSCs.

To address this, investigators have engrafted immunodeficient mice with human HSCs as in vivo animal models for studies of human HSC function (8, 9). These models have been limited by relatively low levels of engraftment and the failure of the engrafted human HSCs to differentiate into fully functional T cells, B cells, myeloid cells, and dendritic cells (DCs). To address these difficulties, we previously developed the NOD-scid genetic model for human hematolymphoid engraftment (10). In contrast to C.B-17-scid mice, NOD-scid mice have relatively low levels of NK cell cytotoxic activity, an absence of hemolytic complement, and functionally immature macrophages (10, 11, 12, 13). Because human HSCs engraft in NOD-scid mice at much higher levels than in C.B-17-scid mice, NOD-scid mice have become the standard model in the stem cell research community for evaluation of in vivo human HSC function (14).

However, HSC function in NOD-scid mice is limited by remaining NK cell activity and a relatively short life span due to the early occurrence of thymic lymphomas. To begin to address these limitations, we developed NOD-scid mice homozygous for a targeted mutation in the β₂-microglobulin structural gene (NOD-scid B2m^null mice). These mice are severely deficient in NK cell activity and support higher levels of human HSC engraftment than do NOD-scid mice (15, 16, 17). However, long term studies in NOD-scid B2m^null mice have been constrained by a markedly shortened life span due to accelerated thymic lymphomagenesis (15). Alternative immunodeficient NOD mouse models that have been developed include NOD mice bearing a targeted mutation at the recombination activation gene 1 (NOD-Rag1^null) (18) and NOD-Rag1^null mice bearing a targeted mutation at the perforin (Prf1) locus (NOD-Rag1^nullPrf1^null) (19). NOD-Rag1^nullPrf1^null mice have an increased life span as compared with NOD-scid mice, and they support relatively high levels of human hematolymphoid engraftment (19). Although improved human HSC engraftment was observed in each of these newer immunodeficient NOD models, human HSC failed to differentiate into mature human lymphoid and myeloid cells.

Recently, new murine mouse models suitable for human HSC engraftment have been reported that lack a functional common IL-2 γ-chain (IL2Rγ^null) (20, 21, 22). In humans, a deficiency of IL-2 receptor γ-chain causes X-linked SCID (23). This molecule is indispensable for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 high affinity binding and signaling (24, 25). IL-2Rγ-chain deficiency blocks NK cell development and results in additional defects in innate immunity (26).

We now report the generation and characterization of NOD-scid IL2Rγ^null mice developed in our laboratories. These animals are of unusual interest because they lack mature lymphocytes and NK cells, express other severe impairments in innate immunity, do not develop thymic lymphomas, and are long-lived. Moreover, cytokine-mobilized human peripheral blood stem cells engraft at high levels in NOD-scid IL2Rγ^null mice and develop into human CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells, Ig⁺B cells, myeloid cells, NK cells, and plasmacytoid DCs.

NOD/LtJ (abbreviated as NOD), C57BL/6J, NOD.CB17-Prkdc^scid (abbreviated as NOD-scid), and B6.129S7-Rag1^tm1Mom/J (abbreviated as B6-Rag1^null) mice were raised in our research colony at The Jackson Laboratory under specific pathogen-free conditions as previously described (10, 18). B6.129S4-IL2rg^tmWjl/J mice (abbreviated as B6-IL2Rγ^null) were obtained from the Animal Resources colony at The Jackson Laboratory. The NOD.Cg-Prkdc^scid IL2rg^tmWjl/Sz (abbreviated as NOD-scid IL2 Rγ^null) mouse genetic stock was developed by first crossing NOD-scid females with B6.129S4-IL2Rγ^tm1Wjl/J males. The IL2Rγ mutation is X-linked. (NOD × B6) F₁ +/scid IL2Rγ^null hemizygous males were backcrossed with NOD-scid females. After two backcross generations, females homozygous for scid and heterozygous for the disrupted IL2Rγ gene were identified by flow cytometry and PCR. Homozygosity for scid was determined by flow cytometric analysis of blood for the absence of CD3⁺ T cells and Ig⁺ B cells (10, 15) and confirmed by PCR analyses of tail DNA (27). Heterozygosity for the IL2Rγ^null allele was determined by PCR for the neomycin resistance gene (neo) (http://jaxmice.jax.org/pubcgi/protocols/protocols.sh?objtype=protocol&protocol_id=701).

After backcrossing the IL2Rγ^null allele for 8 generations onto the NOD-scid background, NOD.Cg-Prkdc^scid IL2Rγ^Tm1Wjl/Sz (abbreviated as NOD-scid IL2Rγ⁺/^null) females were crossed with NOD-scid IL2Rγ^null/Y males. NOD-scid IL2Rγ^null female and NOD-scid IL2Rγ^null/Y male offspring were identified by real time quantitative PCR (28) and confirmed by flow cytometric validation of the absence of IL-2Rγ-chain expression on peripheral blood myeloid cells, as described below, using PE-conjugated rat anti-mouse IL2Rγ common chain mAb, clone TUGm2 (BD Biosciences). The NOD-scid IL2Rγ^null females and NOD-scid IL2Rγ^null/Y males were intercrossed. This genetic stock was maintained in positive individually ventilated racks by sib mating NOD-scid IL2Rγ^null mice in a barrier mouse room. After weaning, the colony was expanded and housed in a conventional specific pathogen-free mouse room maintained at 68°F with 14:10 h of light:dark cycle. Mice were fed National Institutes of Health 31 6% fat diet (Purina Mills International) and received acidified (HCl; pH 2.8–3.2) water containing trimethoprim-sulfamethoxazole (Goldline Laboratories) ad libitum on 7 consecutive days every other week as described (19). All experiments were conducted following the guidelines of the Institutional Animal Care and Use Committees at The Jackson Laboratory, St. Jude Children’s Hospital, and University of Massachusetts Medical School.

A group of NOD-scid IL2Rγ^null mice (29 females and 5 males) were monitored weekly. Moribund mice were necropsied as described (19).

Preparation of spleen cells for single and multilabel flow cytometric analysis was performed as previously described (18, 19). The following mAbs against mouse leukocyte markers were purchased from BD Biosciences as FITC, PE, or allophycocyanin (AP) conjugates: anti-CD3, clone 145-2C11; anti-CD4, clone RM4-5; anti-Igκ L chain, clone 187.1; anti-pan granulocyte (Gr-1), clone RB6-8C5; and anti-pan NK cell, clone DX-5. Additional mAbs were generated as hybridoma tissue culture supernatants or as ascites, precipitated with ammonium sulfate, purified by size exclusion chromatography, and labeled with PE, FITC, or AP. These mAbs included: anti-CD122 (IL-2R β-chain), clone TMβ1; anti-B220, clone RA3-6B2; anti-Mac-1, clone M1/70; anti-Ter 119; anti-LGL-1 (Ly49G2), clone 4D11; anti-MHC class I (H-2K^d), clone SF1–1.1; and anti-MHC class II (IA^k,g7), clone 10-2.16. All samples were incubated with propidium iodide to allow exclusion of dead cells from the final analysis. Nucleated cells were selected by light scatter. Flow cytometric analysis was conducted on 10⁴-2 × 10⁴ viable leukocytes using a FACSCalibur (BD Biosciences).

Blood was collected from the retro-orbital venous plexus of 8- to 10-wk-old mice using EDTA-coated capillary tubes (Drummond Scientific). Erythrocytes, leukocytes, and platelets were analyzed using an ADVIA 120 Hematology System (Bayer) as previously described (29). Mice were euthanized in an atmosphere of 100% CO₂ and necropsied. Axillary, brachial, cervical, and inguinal lymph nodes, thymus, spleen, kidney, liver, brain, spinal cord, lungs, pancreas, and femurs were fixed in Bouin’s fixative (30), embedded in paraffin, sectioned at 5 μm, and stained with Mayer’s H&E. Immunocytochemical localization of human lymphoid cells in spleens of engrafted mice was conducted by staining with anti-human CD45, clone H130 (BD Pharmingen) using a three-step avidin-biotin M.O.M. Elite kit (Vector Laboratories) (15).

Mouse Ig was quantified by ELISA as previously described (15). Goat anti-mouse Ig (heavy and L chain specific; Southern Biotechnology Associates) was used as the first layer, and alkaline phosphatase-labeled Ab to mouse κ-chain (Southern Biotechnology Associates) was used as the second layer. Total IgG (Calbiochem) standard curves were run with each assay and Ig levels were determined from the curves. Optical densities of ELISA samples were read at 405 nm on an EL312e ELISA plate reader (Bio-Tek Instruments) (15).

Splenic NK cell cytotoxic activity was determined as described (10). Briefly, mice were injected i.p. with 100 μg of polyinosinic-polycytidylic acid (poly(I:C); Sigma-Aldrich) 36 h before recovery of spleen cells. ⁵¹Cr-labeled YAC-1 cells (American Type Culture Collection) were used as targets. Various E:T ratios were set up in triplicate in V-bottom 96-well microtiter plates. After a 4-h incubation of effector and target cells at 37°C, supernatants were recovered, and the amount of released radioactivity was quantified using a gamma counter. The percent specific ⁵¹Cr release was calculated as described (10).

DC cultures for each strain were established from BM of individual mice as previously described (31). Forty-eight hours after initiation of culture, nonadherent cells were suspended by gentle swirling, and one-half the medium containing nonadherent cells was discarded and replaced with fresh medium supplemented with 500 U/ml GM-CSF and 1000 U/ml IL-4. On day 4, nonadherent cells were suspended as described above, and fresh medium supplemented with cytokines was added as on day 2. At this time, an agonist anti-CD40 mAb (clone HM40-3; BD Biosciences), 5 μg/ml, was added; 2 days later, on day 6 of culture, the nonadherent cells were harvested, and the supernatants were saved for cytokine ELISA. Mouse IFN-γ and IL-12p40 was quantified according to the manufacturer’s instructions using the R&D Systems DuoSet ELISA Development System (IL-12p40) and R&D Systems Quantikine M (IFN-γ). The nonadherent cells were counted and then analyzed by flow cytometry. FITC-conjugated anti-CD86 mAb (clone GL1), PE-conjugated anti-CD11c mAb (clone HL3), PerCP-conjugated anti-CD11b mAb (clone M1/70), and AP-conjugated anti-TNF-α (clone MP6-XT22) were obtained from BD Biosciences. Isotype controls (BD Biosciences) included FITC-conjugated rat IgG1κ (clone R3-34), PE-conjugated rat IgG1 (clone R3-34), PerCP-conjugated rat IgG2bκ (clone A95-1), and AP-conjugated rat IgG1λ (clone A110-1). Four-color flow cytometric analyses of cultured DCs were performed as previously described (31). Stained cells were washed, fixed in 1% paraformaldehyde-PBS, and analyzed using a FACScan flow cytometer (BD Biosciences). Viable lymphoid cells were gated according to their light-scattering properties, and 2.5–5.0 × 10⁴ events were acquired for each analysis.

NOD-scid IL2Rγ^null and control NOD-scid mice were exposed to varying doses of whole body irradiation at a rate of 139 cGy/min from a Shepard Mark I irradiator containing ¹³⁷Cs (J. L. Shepard). The mice were examined daily and euthanized by CO₂ asphyxiation when moribund. Surviving mice were euthanized and necropsied, as described above, at 8 wk after irradiation. H&E-stained sections of thymi and spleens were fixed and examined for the presence of lymphomas.

To compare the abilities of NOD-scid and NOD-scid IL2Rγ^null mice to support human HSC engraftment, experiments were conducted with mobilized HSC from individual donors. Peripheral blood cells from healthy donors were obtained after G-CSF administration (10 μg/kg/day injected s.c. (Neupogen; Amgen, Thousand Oaks, CA) for 4 days). A single leukaphoresis was performed on day 5 using a Cobe Spectra (Cobe). The donors gave informed written consent, and the St. Jude Children’s Hospital Institutional Review Board approved the study. CD34⁺ cells were selected using the Miltenyi CliniMACS system (32). The purity of the CD34⁺ cells after isolation was >90%. NOD-scid IL2Rγ^null and NOD-scid control mice were irradiated with 325 cGy and injected via the tail vein with 7 × 10⁵ CD34⁺ mobilized human HSC in 0.3 ml of medium 24 h after irradiation. The mice were monitored and treated according to institutional guidelines. A cohort of engrafted NOD-scid IL2Rγ^null and NOD-scid mice received Fc-IL7 fusion protein (EMD Lexigen Research Center) injected via the tail vein at a dose of 20 μg once a wk starting 1 wk after the CD34⁺ cell infusion and continuing until the mice were sacrificed.

The Fc-IL7 fusion protein was expressed from a gene construct in which the coding sequence of a signal peptide was fused to the genomic coding sequence of the Fc fragment of human Ig. The coding sequence of the human IL-7 was fused in frame to the carboxyl terminus of the Fc (33). At 8–10 wk after the human CD34⁺ cell injection, blood was collected via cardiac puncture. The femurs, tibias, and spleens were recovered, cell suspensions were prepared, and the relative percentages of human cells determined by flow cytometric analysis of human CD45 expression on leukocytes. After lysis of RBC, nucleated cells were washed with PBS containing 5% FBS. Samples containing 1 × 10⁶ cells were incubated for 30 min at 4°C with mouse anti-human Abs against the following Ags: CD45, CD3, CD4, CD8, CD14, CD19, CD20, CD56, CD133, BDCA-2, IgM, TCRαβ, and TCRγδ (ΒD Biosciences). For analyses of human leukocyte populations in engrafted mice, peripheral blood, splenocytes, and thymocytes of engrafted mice were gated on human CD45⁺ cells. Mice exhibiting <1% human CD45⁺ cells in the bone marrow were considered nonengrafted and were excluded from the final analysis.

DNA was isolated from single-cell suspensions from spleen and thymus of engrafted mice using the QIAamp DNA mini Kit (Qiagen) following the manufacturer’s instructions. RNA was purified from the same samples with the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions.

Relative sjTREC levels were determined as previously described (34). In brief, real time PCR was performed on 200 ng of DNA using an ABI 7700 system (Applied Biosystems). All samples were studied in duplicate reactions, using primers and conditions as described (34). The Cα constant region was used as an internal control to normalize for input DNA. A standard curve was generated by cloning the sjTREC fragment in pCR 2.1 TOPO vector using the TOPO TA Cloning Kit (Invitrogen). The standard curve and TREC values were analyzed by Sequence Detector software. The numbers of TREC molecules in the samples was calculated as numbers of copies per 10⁵ cells.

The CDR3 size distribution of 27 distinct TCR Vβ families was determined by RT-PCR as previously described (35). In brief, RNA was extracted and cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen). PCR was performed with a forward primer specific for one of the TCR Vβ families along with a constant Cβ reverse primer labeled with fluorescent FAM (36). RT-PCR products were analyzed on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems), using GeneScan software. The normal TCR Vβ CDR3 size was characterized by a Gaussian distribution, containing 8–10 peaks for each Vβ subfamily. The overall complexity of TCR Vβ subfamilies was determined by spectratype scoring as previously described (37).

To examine human αβ T cell development in mice, we established TCR Cβ real time RT-PCR. Total RNA was extracted with an RNeasy Mini Kit (Qiagen). cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen) according to manufacturer’s instructions. PCR assays were performed with Cβ forward primer (5′-CAGCCCGCCCTCAATGACT-3′) and Cβ reverse primer (5′-ACAGGACCCCTTGCTGGTA-3′) on an ABI Prism 7700 Sequence Detector (Applied Biosystems). All samples were measured in duplicate PCR. Human GAPDH mRNA was used as the endogenous control to normalize for input RNA. Experimental samples and data points for the standard curves were assayed in duplicate.

Leukocytes were aseptically prepared as single-cell suspensions from the spleens of engrafted mice. Cells were washed in HBSS after treatment with erythrocyte lysis buffer and then suspended in RPMI complete medium supplemented with 10% (v/v) heat-inactivated FBS and 50 μM 2-ME. The spleen cells were cultured at 2 × 10⁶/ml (100 μl/well), at 37°C in a 5% CO₂, 95% humidity atmosphere in round-bottom 96-well microtiter plates (Costar), with medium alone or with medium containing 1 μg/ml PHA or 1 μg/ml SPE-C (Toxin Technology). After incubation for 72 h, the cultures were pulsed with 1 μCi/well [³H]thymidine (specific activity, 6.7 Ci/mmol) and incubated for 6 h before harvesting. The cells were harvested onto glass fiber filters, and the amounts of [³H]thymidine incorporated were measured using a β-scintillation counter (MatrixTM96 β counter; Packard Instruments). Samples were assayed in triplicate, and the data are presented as mean cpm [³H]thymidine uptake ± SD.

Human serum IgG and IgM levels in engrafted mice were determined according to the manufacturer’s instructions using ELISA kits from BETHYL Laboratories.

All measures of variance are presented as SEM. Significance of difference of independent means was assumed for p values of <0.05. Statistical comparisons of survival in NOD-scid IL2Rγ^null mice were performed using the method of Kaplan and Meier (38). The equality of survival distributions for NOD-scid and NOD-scid IL2Rγ^null mice was tested using the log rank statistic (39).

The IL-2R γ-chain is required for signaling through six discrete cytokine receptors on multiple hemopoietic cell populations (24, 25). We hypothesized that loss of IL-2Rγ-chain function would decrease innate immunity in NOD-scid mice and enhance human HSC engraftment. We backcrossed the IL2Rγ^null allele onto NOD-scid mice, a commonly used mouse strain for human HSC engraftment (14, 40). Before testing human HSC engraftment in NOD-scid IL2Rγ^null mice, we first defined the phenotypic, histological, and functional properties of their lymphohemopoietic system.

Histological examination of lymphoid tissues from five pairs of 3-mo-old NOD^+/+, NOD-scid, and NOD-scid IL2Rγ^null mice was conducted. Fig. 1 shows representative histology of a thymus, spleen, and lymph node of each of the three strains examined. NOD+/+ lymphoid tissues showed normal structure (Fig. 1, A, D, G, J, M, and P). In contrast, severe depletion of lymphoid cells was evident in lymphoid tissues from NOD-scid and NOD-scid IL2Rγ^null mice. The thymi from NOD-scid mice contained a small population of mononuclear cells (Fig. 1, B and E), some of which had mitotic figures and are likely to be premalignant thymic lymphoma cells (not shown). In contrast, thymi from NOD-scid IL2Rγ^null mice consisted mostly of stromal cells with sporadic cystic structures (asterisks) (Fig. 1, C and F). Splenic follicles in NOD-scid mice were hypoplastic but were clearly evident (arrows) (Fig. 1, H and K). In contrast, follicles were not detected in the spleens of NOD-scid IL2Rγ^null mice (Fig. 1, I and L). Although lymph nodes in both NOD-scid (Fig. 1, N and Q) and NOD-scid IL2Rγ^null mice (Fig. 1, O and R) were hypocelluar, NOD-scid IL2Rγ^null lymph nodes were markedly smaller than NOD-scid lymph nodes. There were no marked histological abnormalities in kidney, liver, lung, brain, spinal cord, pancreas, or femurs from NOD-scid IL2Rγ^null mice (data not shown).

NOD-scid mice have a median life span of only 37 wk due to the early development of thymic lymphomas (10). In contrast, 29 of 34 NOD-scid IL2Rγ^null mice remained alive at 59–95 wk of age (median survival time, >89 wk; p < 0.001, Fig. 2). Five NOD-scid IL2Rγ^null mice that appeared moribund at ages ranging from 47 to 68 wk of age were necropsied. None of these mice had thymic lymphomas. Two of the five mice at 47 and 61 wk of age, respectively, had lymphomas evident in the spleen. One mouse had a mammary adenocarcinoma. Two of these mice had no detectable neoplasms, and the cause of death was undetermined. All of the remaining 29 NOD-scid IL2Rγ^null mice had no gross lesions and appeared healthy. Absence of thymic lymphomas in NOD-scid IL2Rγ^null mice is shown in Fig. 3. By 40 wk of age, the majority of NOD-scid mice die with thymic lymphomas (Fig. 3, A and B). In contrast, NOD-scid IL2Rγ^null mice are highly resistant to thymic lymphomas. A NOD-scid IL2Rγ^null mouse euthanized at 68 wk of age (Fig. 3, C and D) shows no evidence of lymphoma.

Engraftment of scid mice by human HSC requires conditioning by irradiation or myeloablative drugs (14). Because irradiation accelerates the development of thymic lymphomas in NOD-scid mice, long term human HSC engraftment studies are difficult to perform (41, 42, 43). To determine the effect of the IL2Rγ^null allele on radiation-induced thymic lymphomagenesis, groups of 6-wk-old NOD-scid and NOD-scid IL2Rγ^null mice were irradiated with 200–450 cGy and monitored for 8 wk. As shown in Table I, NOD-scid mice survived irradiation doses of up to 400 cGy, but all of these animals became moribund and were sacrificed following administration of 450 cGy. In contrast, the NOD-scid IL2Rγ^null mice were slightly more radiosensitive, and 100% of these mice became moribund and were sacrificed following exposure to 400 cGy. All NOD-scid IL2Rγ^null mice irradiated with doses below 400 cGy survived during the 8-wk observation period. The NOD-scid and NOD-scid IL2Rγ^null mice that survived irradiation were necropsied at 8 wk following treatment to determine effects of radiation on thymic lymphoma development. Two of 25 irradiated NOD-scid mice had thymic lymphomas (Fig. 3, E and F) and many of the other NOD-scid thymi exhibited numerous mitotic figures (not shown), suggesting premalignant changes. In contrast, none of the NOD-scid IL2Rγ^null thymi from the irradiated animals showed lymphoma development (Fig. 3, G and H).

Multiple hemopoietic cell populations require signaling through the IL2Rγ-chain for development and function (24). To determine the effects of loss of the IL2Rγ-chain, particularly in NK cells that constrain human HSC engraftment in scid mice, we conducted flow cytometric analyses. The phenotypic profiles of spleen cell populations of 3- to 4-mo-old NOD-scid IL2Rγ^null and NOD-scid control mice are shown in Table II. NOD/Lt+/+ mice were included as a positive control for the staining specificity of Abs against cell surface Ags on mature T and B cells (data not shown). There was a 2-fold reduction in nucleated spleen cell numbers in NOD-scid IL2Rγ^null mice (4.4 ± 0.3 × 10⁶⁾ compared with NOD-scid mice (8.1 ± 1.0 × 10⁶; p < 0.01). As expected, both NOD-scid IL2Rγ^null and NOD-scid spleens were deficient in mature (CD3⁺CD4⁺ and CD3⁺CD8⁺) T cells and in mature B220⁺Igκ⁺ B cells. NOD-scid IL2Rγ^null mice had a significant reduction in percentages of B220⁺Igκ⁻ immature B cells compared with NOD-scid mice. Of particular interest was the finding that splenocytes from NOD-scid IL2Rγ^null mice were deficient in cells expressing NK cell markers (LGL⁺CD122⁺ cells or LGL⁺DX5⁺ cells) compared with NOD-scid mice (p < 0.05). Percentages of Gr-1⁺Mac-1⁺ monomyeloid cells were significantly elevated in NOD-scid IL2Rγ^null mice compared with NOD-scid mice (p < 0.05). Almost all nucleated cells in spleens of mice of both genotypes expressed H-2K^d (MHC class I). However, consistent with the lack of mature B cells, both NOD-scid IL2Rγ^null mice and NOD-scid mice had low percentages of cells that expressed I-A^g7 (MHC class II).

Examination of peripheral blood cell populations from five pairs of male NOD-scid IL2Rγ^null and NOD-scid mice at 10 wk of age showed a significant decrease in peripheral leukocyte counts in NOD-scid IL2Rγ^null mice (1.55 ± 0.2 × 10⁶/ml) compared with NOD-scid mice (2.25 ± 0.1 × 10⁶/ml; p < 0.05). Packed cell volumes in NOD-scid IL2Rγ^null mice (37.9 ± 0.5%) were slightly lower than in NOD-scid mice (39.2 ± 0.3%; p < 0.05). There were no significant differences between NOD-scid IL2Rγ^null and NOD-scid mice in differential leukocyte counts, erythrocyte counts, or platelet counts (data not shown).

Because of evidence of functional lymphocytes in both T and B cell compartments in some leaky scid mice (44) and a concern that leakiness may affect human HSC engraftment, we determined levels of serum Ig in 11 female and 12 male NOD-scid IL2Rγ^null mice at 394–426 days of age. There was no detectable Ig (<0.1 μg/ml) in any of these serum samples. Control pooled sera from three NOD+/+ mice tested at 11 wk of age had 496 μg/ml Ig. Our previous study showed that ∼7% of NOD-scid mice expressed >1 μg/ml serum Ig at 5–7 mo of age (10).

Host NK cell activity is a major obstacle to human HSC engraftment (10, 14, 16, 19). Based on our flow cytometry data showing an absence of cells expressing NK cell surface markers (Table II and Fig. 4,A), NOD-scid IL2Rγ^null mice would be expected to lack NK cell activity. We observed that spleen cells from poly(I:C)-stimulated NOD-scid IL2Rγ^null mice tested at 6–8 wk of age exhibited extremely low levels of NK cell cytotoxic activity (Fig. 4 B). In contrast, C57BL/6-Rag1^null spleen cells included as a positive control for NK cell cytotoxic activity (18) showed greatly elevated NK cell cytotoxic activity.

Maturation of DC populations in NOD-scid IL2Rγ^null mice were analyzed because NOD mice have previously been shown to have abnormalities in DC populations (31) and DC activity in murine BM might limit human HSC engraftment. BM DC cultures were established from NOD-scid IL2Rγ^null and control mice. As shown in Fig. 5, DC cultures from NOD-scid IL2Rγ^null mice generate abnormally low percentages of TNF-α⁺ and CD86⁺ DCs. Analyses of culture supernatants revealed that NOD-scid IL2Rγ^null DC cultures failed to produce either IL-12p40 or IFN-γ.

To determine the ability of NOD-scid IL2Rγ^null mice to support engraftment of human HSC, groups of NOD-scid IL2Rγ^null and NOD-scid mice were sublethally irradiated and injected with human CD34⁺-mobilized stem cells. Preliminary pilot experiments revealed that NOD-scid IL2Rγ^null mice supported heightened levels of human CD34⁺ HSC engraftment compared with NOD-scid mice (data not shown). To evaluate human HSC engraftment in NOD-scid IL2Rγ^null mice and to determine effects of a human Fc-IL7 fusion protein on lymphocyte development, CD34⁺ cells (7 × 10⁵) were injected into 17 NOD-scid IL2Rγ^null and 16 NOD-scid mice. Mice with >1% human CD45⁺ cells in the bone marrow were considered as engrafted. Four of the NOD-scid IL2Rγ^null mice and four of the NOD-scid received weekly injections of human Fc-IL7 fusion protein starting at the time of human HSC injection. All of the 13 NOD-scid IL2Rγ^null mice that did not receive Fc-IL7 and 3 of 4 NOD-scid IL2Rγ^null mice that were treated with this cytokine showed >1% human CD45⁺ hemopoietic cell engraftment in the BM at 10 wk following human HSC injection. In contrast, only 8 of 13 of the untreated NOD-scid control mice supported engraftment. None of the four Fc-IL7-treated NOD-scid mice supported engraftment. Percentages of human cells in the BM of the engrafted NOD-scid IL2Rγ^null mice were 5- to 6-fold higher than in the BM of engrafted NOD-scid mice (Table III). Fc-IL7 treatment of NOD-scid IL2Rγ^null mice did not increase levels of human CD45⁺ cell engraftment in the BM (Table III).

Flow cytometric analyses of the BM from the NOD-scid IL2Rγ^null mice regardless of treatment with Fc-IL7 showed multilineage engraftment (Fig. 6). A high percentage of human CD45⁺cells in the BM were CD19⁺CD10⁺ immature B cells (Fig. 6,A). Approximately one-third of these CD19⁺ cells were mature as defined by coexpression of CD19 and IgM (Fig. 6,B). The BM from these engrafted NOD-scid IL2Rγ^null mice also contained human CD56⁺ NK cells, CD14⁺ myeloid cells (Fig. 6,C), BDCA-2⁺ plasmacytoid cells, and CD133⁺ stem cells (Fig. 6 D).

Human CD45⁺ cells comprised >50% of the spleen cells of engrafted NOD-scid IL2Rγ^null mice (Table III). In contrast, only 5.2% of the spleen cells of engrafted NOD-scid mice were of human origin. Analyses of human CD45⁺ cell populations in the spleens of engrafted NOD-scid IL2Rγ^null mice not treated with Fc-IL7 showed high levels of CD20⁺ B cells and lower levels of CD3 T cells (Fig. 7,A). In contrast, human CD45⁺ cells in the spleens of engrafted NOD-scid IL2Rγ^null mice treated with Fc-IL7 included high percentages of CD3⁺ T cells and lower percentages of human CD20⁺ B cells (Fig. 7,B). Most of the human cells in the spleens of engrafted NOD-scid were CD19⁺ B cells (data not shown). Immunocytochemical staining for expression of human CD45 showed that the human leukocytes in the spleens of engrafted NOD-scid IL2Rγ^null mice were present in follicular structures regardless of whether the mice received Fc-IL7 (Fig. 8, A–D). Human lymphocytes were present in the periarteriolar lymphoid sheaths (PALS) of spleens in the engrafted Fc-IL7-treated NOD-scid IL2Rγ^null mice (Fig. 8, A and B) but there was only limited engraftment of PALS in the absence of Fc-IL7 treatment (Fig. 8, C and D). Lymphoid follicles were not present in the spleens of NOD-scid mice (Fig. 8, E and F).

To determine whether Ig-secreting plasma cells developed in engrafted mice, serum was prepared from one Fc-IL7-treated NOD-scid IL2Rγ^null mouse and three untreated NOD-scid IL2Rγ^null mice 10 wk after engraftment with human HSC. Mean human IgG levels for the Fc-IL7-treated and untreated mice were 84.7 and 0.6 ± 0.3 μg/ml, respectively. Mean human IgM levels for the Fc-IL7-treated and untreated mice were 22.1 and 5.5 ± 2.2 μg/ml, respectively.

Examination of the thymus of Fc-IL7-treated NOD-scid IL2Rγ^null mice revealed high percentages of CD4⁺CD8⁺ cells (Fig. 9,A). CD45⁺ cells in the peripheral blood of these mice included 73% CD3⁺ T cells and 3.6% CD56⁺ NK cells (Fig. 9,B); 34% of CD45⁺ cells in the peripheral blood expressed CD4, whereas 55% expressed CD8 (Fig. 9,C). Most of these human peripheral T cells (45%) expressed TCRαβ, whereas a smaller percentage (22%) expressed TCRγδ (Fig. 9 D). Fewer than 1% of thymocytes of engrafted NOD-scid (including those treated with Fc-IL7) mice expressed human CD45 (data not shown).

To evaluate the complexity of the T cells that developed in engrafted mice, samples of tissues were subjected to several different types of analysis. We first analyzed TCR Cβ expression via real time PCR (Table IV). NOD-scid IL2Rγ^null mice treated with Fc-IL7 had substantially greater amounts of PCR product for TCR Cβ than NOD-scid IL2Rγ^null mice not treated with the cytokine (Table IV). This is consistent with the finding of increased numbers of human CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells in Fc-IL7-treated NOD-scid IL2Rγ^null mice (Fig. 7). In contrast, NOD-scid mice treated with Fc-IL7 had negligible levels of TCR Cβ PCR product (Table IV) and contained <1% human CD3⁺ T cells (data not shown).

The second tool used to evaluate human T cell development was TREC analysis. Formation of TRECs results from intrathymic TCR rearrangement (45). Quantitation of the amount of TRECs for each thymus sample provides an estimate of TCR rearrangement within the thymus. In the Fc-IL7-treated NOD-scid IL2Rγ^null mice, TREC levels were relatively high. In contrast, neither NOD-scid mice treated with cytokine nor untreated NOD-scid IL2Rγ^null mice had detectable TREC levels (Table IV). The third tool used for analysis of human T cell development in engrafted mice was Vβ spectratyping. This PCR-based method determines the length of different TCR CDR3β motifs. The distribution of lengths of PCR products usually follows a Gaussian distribution. Use of multiple different primers to assess different regions of the CDR3 RNA products produces a picture of the complexity of the T cell repertoire and generates a complexity score. A higher score indicates increased T cell repertoire complexity (46, 47, 48). The Fc-IL7-treated NOD-scid IL2Rγ^null mice had a much higher spectratyping complexity score (SCS) than either the Fc-IL7-treated NOD-scid mice or the NOD-scid IL2Rγ^null mice not treated with cytokine (Fig. 10 and Table IV).

Preliminary analysis of the proliferative responses of human T cells measured the ability of spleen cells from an individual engrafted Fc-IL7-treated NOD-scid IL2Rγ^null mouse to proliferate in response to PHA and to the bacterial superantigen SPE-C. As shown in Fig. 11, spleen cells from this engrafted mouse showed a vigorous proliferative response to these T cell mitogens.

We developed the NOD-scid IL2Rγ^null model in this study to test the hypothesis that multiple defects in adaptive and innate immunity caused by the combined effects of 1) the scid mutation, 2) the IL2Rγ ^null allele, and 3) the NOD/Lt strain background would support high levels of human HSC engraftment and multilineage differentiation. Our results confirmed the presence of severe defects in innate immunity that is achieved by combining these three components. NOD-scid IL2Rγ^null mice lack mature T cells, B cells, and NK cells and are “nonleaky” at >1 year of age. Using BM cell cultures, we documented that their stem cells are also unable to generate a functional DC population. Importantly, their longevity is increased greatly over that of other immunodeficient NOD mice we have generated, and this longevity is associated with an absence of thymic lymphoma development.

The increased resistance to thymic lymphoma development and consequent increased life span of NOD-scid IL2Rγ^null mice, beyond 16 mo of age, enables long term experimentation that has not been possible with shorter lived stocks of immunodeficient NOD mice. We have previously reported that NOD-scid mice have a mean life span of 8.5 mo and die with thymic lymphomas (10). The mean life span of NOD-scid B2m^null mice is further reduced to only 6 mo due to the accelerated appearance and progression of thymic lymphomas associated with lowered NK cell activity in these mice (15). Although NOD-scid B2m^null mice support heightened human HSC engraftment compared with NOD-scid mice (15, 16, 17), the rapid development of thymic lymphomas is a major impediment to long term human HSC engraftment experiments.

The development of malignant T cell progenitors within the thymus of immunodeficient NOD mice is associated with a programmed breakdown in the TCRβ selection checkpoint that leads to a defect in early T cell development and the loss of a critical control checkpoint in proliferation (49). Previous attempts to prevent or delay the development of thymic lymphomas have targeted the activation and subsequent genomic reintegration of a NOD mouse-unique endogenous ecotropic provirus (Emv-30), which appears to enhance lymphoma development (50). Genetic elimination of the Emv-30 provirus by production of a genetic stock of NOD-scid Emv-30^null mice slows the growth but does not eliminate thymic lymphomagenesis (51).

Our present data suggest that the development of thymic lymphomas in NOD-scid mice is absolutely dependent on cytokine signaling mediated through the IL2Rγ-chain. This signaling could be mediated through the high affinity IL-2, IL-4, IL-7, IL-9, IL-15, or IL-21 receptors (24, 25). The thymic lymphomas that develop in NOD-scid, NOD-scid B2m^null, and NOD-Rag1^null mice consist of TCR-negative CD4⁻CD8⁻ double-negative or CD4⁺CD8⁺ double-positive thymocytes that express receptors for IL-2 and IL-7 (49, 50, 52) and (L. D. Shultz, unpublished observations), suggesting that signaling through these cytokine receptors may be essential for lymphoma development. Thymocytes from NOD-scid IL2Rγ^null mice are unable to signal through these cytokine receptors because the IL2Rγ-targeted mutation in these mice is a complete null mutation (26). This precludes any possibility of either binding or signaling through any of these high affinity receptors.

Our results confirm and extend the observations of others studying human HSC engraftment in immunodeficient IL2Rγ^null mice. Three research groups have independently reported the production of mice bearing targeted IL-2R common γ-chain mutations (26, 53, 54). We report here our data on the IL2Rγ^null allele targeted by Cao et al. (26) that we have backcrossed onto the NOD/LtSz-scid genetic background. Targeted IL2Rγ mutations have previously been used by two other groups to create immunodeficient mice for engraftment of human HSC. First, Ito et al. (21) backcrossed a truncated IL2Rγ mutation onto the NOD/Shi-scid strain. Although the truncated mutation precludes intracellular signaling (54), the incidence of thymic lymphomas and the life span of these mice have not been reported. In contrast, we observed that the majority of NOD-scid IL2Rγ^null mice survive >16 mo.

Sublethally irradiated NOD/Shi-scid IL2Rγ^null mice injected i.v. with human UMB HSC supported high levels of engraftment and generated functional T cells starting at 13 wk postengraftment (21, 22, 55) and also developed functional CD5⁺ B cells (56).

However, one potential disadvantage of the NOD/Shi-scid IL2Rγ^null model is that the targeted mutation used in these crosses is not a complete null (21, 22). The ability to modulate human lymphocyte development using cytokines such as IL-7 has not been documented in this host and may be difficult as host cells could compete with the developing human cells for binding of the injected cytokine. Second, Traggiai et al. (20) reported the development of BALB/c-Rag2^nullIL2Rγ^null mice using an IL2Rγ^null knockout originally made by DiSanto et al. (53). These mice support high levels of engraftment with human UMB HSC that develop into a functional adaptive immune system (20). This group used a novel route of intrahepatic injection into newborn mice that also enhances human HSC engraftment, making direct comparison of the protocols and mouse hosts difficult. Furthermore, our earlier studies have shown that NOD-scid mice are superior to BALB/c-scid mice in their ability to support human hemolymphoid cell engraftment (14, 57, 58, 59).

Of additional interest is our observation of the lack of NK cell activity in NOD-scid IL2Rγ^null mice. NK cell development and survival is dependent on signaling through IL-2R (26), and our data confirm that NK cells are severely depleted in these mice. In addition, in other NOD-scid mice that we have previously generated and characterized (10, 14, 15, 59), sporadic T and B cell development was observed. This leakiness is enhanced by irradiation conditioning, a requisite for engraftment of human HSC (14, 60). Due to the lack of signaling through the cytokine receptors required for mature T and B cell development, even irradiated NOD-scid IL2Rγ^null mice failed to become leaky and generate mature T or B cells. It has also been reported that NOD/Shi-scid IL2Rγ^null mice generate defective DCs (21). This was based on the low production of IFN-γ following stimulation with LPS. We have extended this observation to document that BM cultures of NOD-scid IL2Rγ^null mice are deficient in their ability to generate mature DC populations as quantified by their expression of CD86 and TNF-α, and by their inability to produce IL-12p40 or IFN-γ.

To investigate the ability of NOD-scid IL2Rγ^null mice to support human HSC engraftment and differentiation, we used purified human CD34⁺ HSC that had been mobilized from healthy donors. We chose to study this human HSC population in NOD-scid IL2Rγ^null mice for two reasons. First, in the clinical setting, autologous stem cell transplantation is becoming a standard clinical treatment for many diseases, including hemopoietic malignancies (5) and autoimmunity (7). Mobilized HSC are much easier to harvest than are BM HSC and are the stem cells of choice at most transplantation centers. Furthermore, the engraftment of mobilized HSC in immunodeficient mice is more difficult to achieve than when bone marrow or UMB is used (61, 62, 63). This provides a stringent test of the ability of NOD-scid IL2Rγ^null mice to support human HSC engraftment and differentiation.

We observed that NOD-scid IL2Rγ^null mice support high levels of engraftment and multilineage differentiation following injection with mobilized human HSC. Such engraftment results in development of human T cells, B cells, myeloid cells, NK cells, plasmacytoid DCs, and CD133⁺ HSC. The generation of human B cells was documented by their coexpression of CD19 and surface Ig and by the presence of human Ig in their serum. We also observed the development of human T cells in HSC engrafted NOD-scid IL2Rγ^null mice. In previous studies with NOD-scid mice (in the presence of a wild-type IL2Rγ gene), it has been difficult to document the development of human T cells in engrafted mice. In contrast, within only 10 wk after engraftment, human T cells were clearly present in the spleen of engrafted mice. Because T cell development is dependent on epithelially derived human IL-7 (64, 65, 66), we injected a cohort of these mice with an Fc-IL7 fusion protein. Cytokine-Ig fusion proteins demonstrated increased half-life in vivo compared with native cytokines (33, 67).

We observed that exogenous Fc-IL7 greatly enhanced the generation of human T cells in the HSC-engrafted NOD-scid IL2Rγ^null mice. The human T cells that were present in the thymus displayed the expected ratios of double-negative, double-positive, and single-positive CD4 and CD8 populations, and high levels of single-positive CD3⁺ human T cells were present in the spleen and the peripheral blood of treated recipients. The PALS of the spleen in Fc-IL7-treated NOD/LtSz-scid IL2Rγ^null mice was populated with human CD45⁺ cells. However, because our Ab staining was restricted to human CD45 expression in these paraffin sections, we have not demonstrated localization of human T and B cells within the PALS.

Increased percentages of splenic CD8⁺ T cells were observed compared with CD4⁺ T cells. Such skewed CD4:CD8 ratios are commonly seen in the early phase of T cell development following BM engraftment (R. Handgretinger, personal observations). The ability to monitor engraftment and differentiation of human cells in the blood at multiple time points facilitates a better understanding of the sequence of development of T cells without having to sacrifice a mouse at each time point.

The human CD3⁺CD4+ and CD3⁺CD8⁺ T cell populations in the peripheral tissues of engrafted NOD-scid IL2Rγ^null mice could have been generated through two pathways. First, an expansion of graft-derived donor T cells via a thymus-independent pathway would lead to T cells in the periphery, as has been observed following the injection of PBMC (57, 68). Any contaminating mature CD3⁺ T cells present in the CD34⁺ HSC preparation would display a limited TCR repertoire diversity and would likely cause xenogeneic graft vs host disease (GVHD). There was no evidence of GVHD in engrafted mice. In addition to this the purity of the CD34⁺ cells after selection was >90% with only 0.3% CD3⁺ T cells, making it unlikely that the populations of T cells in the NOD-scid IL2Rγ^null mice treated with FC-IL7 originated from the graft. Second, the human CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells could have been derived from a thymus-dependent pathway. That the T cells we observed were generated by this pathway is supported by our phenotypic analysis of the human thymocytes. It would be anticipated that these T cells would display a more diverse TCR repertoire and would not cause GVHD. To confirm the phenotypic data in the thymus, we used three additional methods to identify human thymic-dependent T cell development: 1) TCR CDR3β spectratyping to examine the diversity of the TCR repertoire; 2) TCR Cβ real time PCR to quantify human TCRβ expression in T cells; and 3) the TREC assay to quantify human sjTREC produced from recent thymic emigrants. This latter assay can quantify the level of naive human T cells.

Tenweeks after HSC transplantation, we found high levels of human TCRβ molecule expression, sjTREC production, and TCR receptor diversity in Fc-IL7-treated NOD-scid IL2Rγ^null mice. Comparably treated NOD-scid mice had very low responses in all three of these assays. To confirm that these human T cells were functional, we documented in preliminary experiments that they proliferated in response to PHA and streptococcal superantigen SPE-C. These findings suggest that the NOD-scid IL2Rγ^null mouse model not only improved engraftment but also facilitated thymic-dependent T cell differentiation. Although Fc-IL7 treatment supported human T cell development in NOD-scid IL2Rγ^null mice engrafted with human mobilized HSC, cytokine treatment resulted in relatively lowered percentages of human B cells. However, these B cells appear to be capable of Ig secretion as evidenced by the presence of serum IgG and IgM in these engrafted mice. The dose of Fc-IL7 used in the current study may not have been optimal to support both T and B cell development. Previous reports using transgenic mice have suggested that there is a dosage effect of IL-7 on T cell and on B cell development (69, 70, 71). In this study, lymphoid tissues removed from individual engrafted mice were used for multiple measurements that included histology, flow cytometry, cell culture, and RNA isolation. This constrained our ability to conduct determinations of total cellularity of lymphoid organs. Therefore, we did not measure the effect of Fc-IL7 treatment on total numbers of T and B cells. Future quantitative studies investigating the dose and duration of IL-7 on human T and B cell development in these mice are under way.

Our current study focused on the ability of conditioned adult NOD-scid IL2Rγ^null mice to support engraftment with mobilized human CD34⁺ HSC. Engrafted NOD-scid IL2Rγ^null mice exhibited functions that included the presence of human Ig in the serum and the abilities of human T cells to proliferate in response to mitogens and superantigen. We are currently investigating whether systemic humoral or cellular responses can be elicited in engrafted NOD-scid IL2Rγ^null mice. In separate studies, we have found that engraftment of newborn NOD-scid IL2Rγ^null mice with human CD34⁺ cord blood HSC generates multiple human blood components including granulocytes, monocytes, erythrocytes, and platelets as well as phenotypically mature T and B cells. We further documented that these mice engrafted as newborns developed a functional human immune system following engraftment of newborn NOD-scid IL2Rγ^null mice in the absence of exogenous IL-7, as determined by humoral immune responses to OVA (72).

In summary, we describe the generation and characterization of NOD-scid IL2Rγ^null mice and document the ability of cytokine-mobilized human peripheral blood HSC to engraft these mice at high levels. Our preliminary experiments indicate that the engrafted HSC develop into human CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells, Ig⁺B cells, as well as myeloid cells, NK cells, and plasmacytoid DCs. The human T cells that develop following in vivo treatment with an Fc-IL7 fusion protein show proliferative responses following stimulation. These data suggest that NOD-scid IL2Rγ^null mice will be a robust model for in vivo studies of human HSC engraftment, differentiation, and analysis of human immune systems in response to infectious agents, malignant cells, radiation, and drug regimens without putting patients at risk.

We thank David Serreze and Brian Soper for critical review of the manuscript. We are grateful to Allison Ingalls, Mario Otto, and Thasia Leimig for excellent help with the mice. We thank Jim Houston and Marti Holliday for conducting the FACS studies of the engrafted mice.

The authors have no financial conflict of interest.