Engraftment of human hematopoietic stem cells into immunodeficient mice that lack T cells, B cells, and NK cells results in reconstitution of human blood lineage cells, especially B cells, in the recipient mice. However, these humanized mice do not make any significant level of IgG Ab in response to Ag stimulation. In this study, we show that in humanized mice, B cells are immature, and there is a complete deficiency of CD209+ (DC-SIGN) human dendritic cells. These defects can be corrected by expression of human GM-CSF and IL-4 in humanized mice. As a result, these cytokine-treated humanized mice produced significant levels of Ag-specific IgG after immunization, including the production of neutralizing Abs specific for H5N1 avian influenza virus. A significant level of Ag-specific CD4 T cell response was also induced. Thus, we have identified defects in humanized mice and devised approaches to correct these defects such that the platform can be used for studying Ab responses and to generate novel human Abs against virulent pathogens and other clinically relevant targets.
Adoptive transfer of human hematopoietic stem cells (HSCs) into immunodeficient mice that lack T cells, B cells, and NK cells can lead to stable engraftment of human HSCs (1–5). Differentiation of the transferred HSCs gives rise to different lineages of human blood cells in the recipient mice, which are often referred to as humanized mice. Because humanized mice enable in vivo study of human hematopoiesis, infectious diseases (especially those caused by pathogens that only infect human blood lineage cells), and human blood cell diseases (such as leukemia and lymphoma), the platform has significant applications in both basic and translational biomedical research (1, 2, 6).
In humanized mice, human B cells and T cells are the most abundantly reconstituted cell types among all blood lineage cells. Despite this, humanized mice do not make a significant Ab response after immunization with different Ags in the presence of various adjuvants and through various routes (1, 3, 7). For example, SCID mice that were transplanted with human thymus, bone marrow, and skin failed to generate any Ag-specific IgG after immunization with tetanus toxoid (TT) vaccine (7). Although additional transplantation of human lymph nodes into the recipient mice improved Ab response, the level of TT-specific human IgG was still very low (<0.2 μg/ml) (7). When BALB/c-Rag2−/− Il2rg−/− mice were used as recipients of human blood cells, the resulting mice had around 37 μg/ml and 191 μg/ml of circulating human IgM and IgG, respectively, which were increased to 173 μg/ml and 459 μg/ml after TT immunization. However, the level of Ag-specific human IgM was barely detectable, and no Ag-specific IgG was detected (8). When the same BALB/c-Rag2−/− Il2rg−/− recipient mice were treated with recombinant IL-15/IL-15Rα agonist, the total human IgG level increased to ∼700 μg/ml. After TT immunization, TT-specific human IgG was significantly elevated (0.5 IU/ml) compared with the untreated but immunized humanized mice (0.1 IU/ml) (5). Furthermore, when NOD-scid, Il2rg−/− (NSG) mice were engrafted with human HSCs, no Ag-specific human IgG was elicited upon TT immunization, even after transplantation of human fetal liver and thymus (9). Similarly, humanized NOD-Rag1−/− Il2rg−/− mice had only a low level of human IgM (6 μg/ml) and failed to produce any Ag-specific IgG after TT immunization (10). The poor Ab response in humanized mice has significantly limited the potential of the platform for studying human B cell responses to pathogens and generation of Ag-specific human Abs.
Induction of an IgG Ab response requires cognate interactions between APCs and CD4 T cells and between CD4 T cells and B cells. Many factors are thought to contribute to the poor human Ab responses in humanized mice, including poor reconstitution of myeloid APCs, mismatch between human TCR and mouse MHC molecules, deficiency in human cytokines, and block of T and B cell maturation in humanized mice (1, 3, 4, 11, 12). For example, myeloid cells, including dendritic cells (DCs), are poorly reconstituted because of a lack of appropriate human cytokines (11). As a result, CD209+ DCs, which are critical for Ag capture and priming of T cell immune responses (13–15), are completely absent in humanized mice. Similarly, development and maturation of human B cells is blocked in humanized mice, and T cell function is impaired (12). Transgenic expression of human MHC class I or II molecules in the recipient mice has been shown to enhance HLA-restricted cytotoxic T cell responses (6). However, Ab responses in these mice either remain very poor (9, 10) or have not been reported.
We have previously developed a simple and efficient method to enhance human immune cell reconstitution in humanized mice by expressing appropriate human cytokines via hydrodynamic injection of cytokine-encoding plasmids (11). In particular, we found that expression of human GM-CSF and IL-4 significantly increases reconstitution of human myeloid cells, including DCs. In the current study, we show that GM-CSF and IL-4 treatment promotes development of human CD209+ DCs and maturation of human B cells and T cells. Upon immunization, Ag-specific Ab responses are greatly improved in the cytokine-treated humanized mice, including induction of human neutralizing Abs against H5N1 influenza viruses. These findings show that the poor Ab responses in humanized mice are partly due to a lack of human CD209+ DCs and a block in B and T cell maturation attributed to lack of appropriate human cytokines. Provision of these human cytokines helps overcome these defects and improves Ag-specific Ab responses in humanized mice.
Materials and Methods
HSC isolation, construction of humanized mice, and hydrodynamic gene delivery
Human fetal livers (n > 8) were obtained from aborted fetuses at 15–23 wk of gestation, in accordance with the institutional ethical guidelines of the National University Hospital of Singapore. All women gave written informed consent for the donation of their fetal tissue for research. Fetuses were collected within 2 h of the termination of pregnancy. Fetal liver tissue was initially cut into small pieces and digested with 2 mg/ml collagenase VI (prepared in DMEM) for 15 min at 37°C with periodic mixing. A single cell suspension was prepared by passing the digested tissue through a 100-μm cell strainer (BD Biosciences). Umbilical cord blood was obtained from the National Disease Research Interchange or the Singapore Cord Blood Bank. CD34+ cells from both fetal liver and cord blood were purified with the use of a CD34+ selection kit (Stem Cell Technologies, Vancouver, BC, Canada); the purity of CD34+ cells was 90–99%. Viable cells were counted by trypan blue exclusion of dead cells. All cells were isolated under sterile conditions. To construct humanized mice, 1 × 105 to 2 × 105 CD34+ fetal liver or cord blood cells were injected into pups within 48 h of birth intracardially. Ten to twelve weeks after engraftment, mice were analyzed for reconstitution by flow cytometry analysis of peripheral blood. Mice that had 40% or more human leukocyte reconstitution were used in subsequent experiments. Hydrodynamic injection of plasmid DNA was exactly as described (11). Briefly, human GM-CSF and IL-4 genes were cloned separately into pcDNA3.1(+) vector (Invitrogen). Plasmid DNA was purified by Maxi-prep Kit (Qiagen). For hydrodynamic gene delivery, 10- to 12-wk-old humanized mice were injected with 50 μg of each plasmid in a total of 1.8 ml saline within 7 s using a 27-gauge needle. All research with human samples and mice was performed in accordance with institutional guidelines.
Immunization and ELISA
Ten to twelve weeks after engraftment, mice were immunized with TT vaccine (Tetavax; Sanofi Pasteur). Specifically, 10 μl of the vaccine, representing 1/50 of the recommended vaccination dose for an adult human, was mixed with 90 μl aluminum hydroxide gel (Sigma) and then injected i.p. Mice were boosted twice with the same dose 3 wk apart. After immunization, sera were analyzed by human TT-specific IgG ELISA (MP Biomedicals Germany GmbH). Total human IgM and IgG levels were analyzed with human IgM and IgG kit (Bethyl Laboratories). When 4-hydroxy-3-nitrophenyl acetyl–keyhole limpet hemocyanin (NP-KLH) (Bioresearch Technologies, Novato, CA) was used as Ag, 100 μl of 1 mg/ml NP-KLH was mixed with 100 μl aluminum hydroxide gel and used for immunization as above. Blank ELISA plates (Bethyl Laboratories) were coated with NP-KLH, and sera from immunized mice were added. HRP-conjugated anti-human IgG Ab (Bethyl Laboratories) was used as secondary Ab. To induce Ab response to H5N1 avian influenza viruses, humanized mice were immunized twice 2 wk apart by i.p. injection. Each immunization comprises 100 μl of inactivated H5N1 influenza virus (HA titer 28) with 100 μl of adjuvant (Seppic). Sera were collected 14 d after the second immunization by facial vein bleeding. H5N1 influenza viruses used for the immunization were inactivated A/Anhui/1/05, A/Indonesia/CDC669/06, and A/Hong Kong/213/03.
Single-cell preparation, Abs, and flow cytometry
Preparation of mononuclear cells from liver, lung, spleen, and bone marrow of humanized mice was described previously (11). The following Abs were used: CD20 (2H7), CD10 (HI10a), CD5 (UCHT2), CD27 (M-T271), IgM (G20-127), IgD (IA6-2), CD3 (HIT3a), CD4 (RPA-T4), CD40L (TRAP1), CD25 (2A3), CD19 (HIB19), HLA-DR (L243), CD209 (DCN46), CD45 (2D1), CD69 (L78), CD33(WM53) from BD Biosciences; and CD40 (5C3), CD80 (2D10), CD86 (IT2.2), mouse CD45.1 (A20) from BioLegend. Cells were stained with appropriate Abs in 100 μl PBS containing 0.2% BSA and 0.05% sodium azide for 30 min on ice. Flow cytometry was performed on an LSR II flow cytometer using the FACSDiva software (BD, Franklin Lakes, NJ). Ten thousand to 1 million events were collected per sample and analyzed using the FlowJo software.
After the third immunization, single-cell suspensions were prepared from spleens for ELISPOT assay (R&D Systems, Minneapolis, MN). Splenocytes containing 1 × 105 CD3+ cells were plated per well in medium alone or in the presence of either 10 ng/ml of PMA or 0.5 μg/ml of a tetanus toxin peptide (Genscript). Spots were counted using an ImmunoSpot S5 Versa Analyzer (Cellular Technology) and analyzed with ImmunoCapture software (Analysis Software).
Madin–Darby canine kidney (MDCK) cells cultured in 96-well plates were infected with H5N1 subtype viruses. The cells were rinsed with PBS and fixed with 4% paraformaldehyde 24 h postinfection. Fixed cells were incubated with sera from humanized mice (1:50 in PBS) or H5 mAb 2D9 at 37°C for 1 h, rinsed with PBS, and then incubated with a 1:500 dilution of FITC-conjugated rabbit anti-human or anti-mouse IgG (Dako). Ab binding was evaluated by wide-field epifluorescence microscopy (Olympus IX71).
mAb (2D9) and polyclonal sera from humanized mice were serially diluted 2-fold and incubated with 100 virus median tissue culture infectious dose (TCID50) of H5N1 virus isolates for 1 h at room temperature and plated in duplicate onto MDCK cells grown in a 96-well plate. The TCID50 of each H5 strain in MDCK cell culture was determined by the Reed and Muench method. The neutralizing titer was assessed as the highest Ab dilution in which no cytopathic effect was observed by light microscopy.
Data are presented as mean and SEM. Differences between groups were analyzed via Student t test. A p value of <0.05 was considered statistically significant. All calculations were performed using the Origin 8.0 software package.
GM-CSF and IL-4 stimulate production of human CD209+ DCs and maturation of human B and T cells in mice
Humanized mice were constructed by adoptive transfer of CD34+ HSCs isolated from human cord blood or fetal liver into sublethally irradiated NSG newborn pups. Twelve weeks after HSC transfer, PBMCs of recipient mice were stained for human and mouse CD45 to determine the reconstitution level of human leukocytes [% human CD45+ cells/(% human CD45+ cells + % mouse CD45+ cells)]. Mice with 40% or more human leukocyte reconstitution were used in this study. To stimulate human DC reconstitution, humanized mice between 12 and 16 wk of age were hydrodynamically injected with vector plasmid (control) or plasmids encoding human GM-CSF and/or IL-4. Seven days after injection, splenocytes were stained for Abs specific for human CD45, CD11c, and CD209 and analyzed by flow cytometry. The frequency of CD11c+ cells within the human CD45+ leukocyte population was increased significantly in mice treated with GM-CSF (∼17%) or GM-CSF plus IL-4 (∼20%) compared with control mice (∼6%) or IL-4–treated mice (∼7%) (Fig. 1A, 1B). A prominent population of human CD209+ DCs was detected only in GM-CSF plus IL-4–treated mice, reaching ∼30% of CD45+CD11c+ human cells. To investigate whether the cytokine-induced CD209+ DCs are functional, 7 d after injection of GM-CSF and IL-4 plasmids, humanized mice were challenged with LPS. Twenty-four hours later, splenocytes were stained for human CD45, CD209 plus CD40, CD80, CD86, or HLA-DR. Although CD40 expression did not change significantly after LPS stimulation, expression of CD80, CD86, and HLA-DR on CD209+ DCs was significantly upregulated (Fig. 1C). When human CD209+ DCs were purified from peripheral blood and spleen by cell sorting and stimulated with LPS in vitro, a significant level of human IL-712p70 was detected in the culture supernatant (Fig. 1D). These results show that expression of GM-CSF and IL-4 in humanized mice stimulates reconstitution of functional human CD209+ DCs.
Expression of human GM-CSF and IL-4 in humanized mice also has a significant effect on the maturation status of human T and B cells. Seven days after injection of vector plasmid or GM-CSF and IL-4 plasmids, splenocytes from humanized mice were stained for human CD45, CD19 plus CD5, CD40, CD20, HLA-DR, CD10, IgM, CD80, or CD86. After IL-4 or GM-CSF plus IL-4 treatment, the levels of CD40, IgM, and CD86 on CD45+CD19+ B cells were significantly elevated, whereas the levels of CD5 and CD20 were reduced (Fig. 1E, Supplemental Fig. 1A, 1B). The expression of HLA-DR, CD10, and CD80 did not change. The frequency of CD19+CD27+ memory B cells was low (∼1%) in humanized mice without cytokine treatment (Supplemental Fig. 1A). After IL-4 or IL-4 plus GM-CSF treatment, the frequency was increased ∼10-fold. Similarly, the proportion of human CD45+CD3+ T cells that expressed CD25 and the proportion of CD45RA−CD3+ T cells were significantly increased after IL-4 or GM-CSF plus IL-4 treatment (Fig. 1F, Supplemental Fig. 1C, 1D). Notably, most CD3+ T cells that expressed CD25 were CD4+ T cells but not CD8+ T cells (Fig. 1G, 1H), suggesting the development of CD4+CD25+ regulatory T cells in humanized mice. Thus, expression of Il-4 alone or GM-CSF plus IL-4 in humanized mice also stimulates T and B cell maturation.
GM-CSF and IL-4 treatment significantly enhances Ag-specific IgG responses in humanized mice
To determine the effect of GM-CSF and IL-4 treatment on Ag-specific Ab responses, we immunized mice 7 d after injection of vector plasmid or GM-CSF and/or IL-4 plasmids with TT vaccine i.p. three times every 3 wk (Fig. 2A). Two weeks after the third immunization, mice were bled for measuring total human IgM and IgG and TT-specific human IgG in the sera by ELISA. Before immunization, the levels of human IgM were similar (∼120 to ∼170 μg/ml) in humanized mice injected with vector control plasmid and cytokine plasmids (Fig. 2B, left panel). Although after injection of IL-4 or GM-CSF plasmid the levels of human IgG were elevated 4- to 6-fold compared with mice injected with vector plasmid, the absolute levels of IgG remained low (1–13 μg/ml, Fig. 2B, right panel). After immunization, the levels of the total IgM did not change much in mice injected with vector or GM-CSF or IL-4 plasmid alone but were increased ∼2.5-fold in mice injected with GM-CSF and IL-4 plasmids (Fig. 2C, left panel). Similarly, the levels of total IgG did not change significantly in mice injected with vector or GM-CSF plasmid. However, the levels of IgG were increased ∼28-fold in IL-4–treated mice, reaching an average of ∼120 μg/ml (Fig. 2C, middle panel). In GM-CSF and IL-4–treated mice, the levels of IgG were increased 55-fold, reaching ∼700 μg/ml. Furthermore, TT-specific IgG (∼0.14 IU/ml) was readily detected in GM-CSF and IL-4 plasmid injected mice after immunization but not in other groups of mice (Fig. 2C, right panel). Among the serum IgG isotypes, the predominant isotype was IgG1, followed by IgG2 and IgG4 (Supplemental Fig. 2A). These results show that GM-CSF and IL-4 treatment greatly stimulates Ag-specific IgG responses in humanized mice.
GM-CSF and IL-4 treatment promotes B and T cell responses after immunization
To investigate the cellular mechanisms of the enhanced Ab responses in GM-CSF and IL-4–treated mice, we analyzed T and B cell responses in the spleen 2 wk after the third immunization. An immediate difference that was noticed was the enlarged spleens from immunized mice that have been injected with GM-CSF and IL-4 plasmids compared with mice injected with vector, GM-CSF, or IL-4 plasmids (Fig. 3A). Flow cytometry analysis revealed that nearly 90% of cells in the spleen of GM-CSF and IL-4–treated mice were human CD45+ (Supplemental Fig. 2B). The total numbers of human leukocytes (CD45+) in the spleen of GM-CSF and IL-4–treated mice were 52.7 ± 12.5 × 106, representing ∼10-fold increase over the numbers in the spleen of mice injected with vector plasmid (4.1 ± 0.4 × 106), IL-4 plasmid (5.2 ± 2.2 × 106), or GM-CSF plasmid (7 ± 3.1 × 106) (Fig. 3B), correlating with the sizes of the spleens. Within the human CD45+ cells, ∼30% was CD19+ B cells and ∼60% was CD3+ T cells in GM-CSF and IL-4–treated mice (Supplemental Fig. 2C, 2D). In comparison, ∼65% were CD19+ B cells and ∼25% CD3+ T cells in vector plasmid-treated (control) mice. As a result, the number of B cells was increased ∼5 times in the spleen of mice injected with GM-CSF and IL-4 plasmids compared with mice injected with vector plasmid, IL-4 plasmid, or GM-CSF plasmid (Fig. 3B). Even more dramatically, the number of CD4+ T cells in the spleen was increased >12 times in mice injected with GM-CSF and IL-4 plasmids compared with mice injected with vector plasmid, IL-4 plasmid, or GM-CSF plasmid. Although the number of CD8+ T cells was also increased in the spleen of mice injected with GM-CSF and IL-4 plasmids, most splenic T cells were CD4+ T cells (Supplemental Fig. 2D).
We further analyzed B cells in immunized mice by staining splenocytes for human CD45, CD19 plus CD40, IgM, CD86, CD5, CD20, HLA-DR, CD10, or CD80. In mice injected with vector plasmid, the phenotype of CD45+CD19+ human B cells did not change significantly after immunization: 80% of CD19+ human B cells expressed CD5 in humanized mice (Fig. 1E) and vector plasmid-injected humanized mice (Fig. 3C). After immunization, 80% of CD19+ human B cells were CD5− in GM-CSF and IL-4–treated mice (Fig. 3C), indicating an induction of conventional (CD5−) B cells. Furthermore, most B cells in the spleen of GM-CSF and IL-4–treated mice exhibited an elevated expression of CD40, IgM, HLA-DR, CD80, and CD86, but reduced expression of CD19, CD20, and CD10 (Fig. 3C, Supplemental Fig. 2B), suggesting maturation of human B cells. Thus, the enhanced Ag-specific IgG response in GM-CSF and IL-4–treated mice is associated with induction of mature conventional B cells.
We also analyzed T cells in immunized mice by staining splenocytes for human CD45, CD3 plus CD25, CD45RA, CD40L, or HLA-DR. Compared to the vector plasmid-injected humanized mice, CD3 T cells upregulated CD40L and HLA-DR, and a small fraction of the T cells was also positive for CD25 in humanized mice injected with GM-CSF and IL-4 plasmids (Fig. 3D, Supplemental Fig. 2D), suggesting that T cells were further activated after immunization. The proportion of CD45RA− T cells also increased significantly in cytokine-treated mice. HLA-DR was upregulated in both CD4+ and CD8+ T cells (Fig. 3E, 3F). When splenocytes were stimulated with a dominant CD4 peptide (QYIKANSKFIGITE) from tetanus toxin, no IFN-γ or IL-4 secretion was detected by ELISPOT assay in vector plasmid-injected (control) mice. In contrast, TT-specific IFN-γ– and IL-4–secreting T cells were readily detected in the splenocytes of GM-CSF and IL-4–treated mice, reaching 1.0 ± 0.2% and 1.8 ± 0.5%, respectively (Fig. 3G). As controls, no IFN-γ or IL-4 secretion was detected without TT peptide stimulation and much higher frequencies of IFN-γ– or IL-4–secreting T cells were detected after PAM stimulation. These results show that GM-CSF and IL-4 stimulate T cell maturation and Ag-specific responses in humanized mice.
To distinguish the effect of cytokines versus immunization on T and B cell numbers, we analyzed humanized mice without immunization 9 wk after injection with control plasmid, GM-CSF, and/or IL-4 plasmids. The numbers of human CD45+ leukocytes in the spleens were increased slightly in IL-4–treated (5.7 ± 0.7 × 106) and GM-CSF plus IL-4–treated (6.5 ± 0.8 × 106) mice compared with control mice (4.5 ± 0.4 × 106) (Supplemental Fig. 3). The numbers of CD19+ B cells in the spleens did not change significantly after cytokine treatment. In contrast, the number of CD3+ T cells was increased ∼2-fold after IL-4 or GM-CSF plus IL-4 treatment. Most of the increase in total T cell numbers was due to increase in CD4+ T cells (∼2.5 times). Because 9 wk after immunization the numbers of CD4+ T cells increased 28-fold in GM-CSF plus IL-4–treated mice compared with control mice (Fig. 3B), the observed effects are primarily due to the combination of cytokine treatment and immunization.
GM-CSF and IL-4–treated mice are capable of generating H5N1 virus-neutralizing Abs
To test whether GM-CSF and IL-4–treated mice produce human Ab responses to other Ags, we used NP-KLH, a commonly used model Ag, and inactivated avian influenza virus H5N1. Cytokine-treated humanized mice were immunized three times at 3-wk intervals with NP-KLH, and sera were measured for total and NP-specific human IgM and/or IgG. Immunization of GM-CSF and IL-4–treated humanized mice with NP-KLH also led to elevated levels of total human IgM (∼445 μg/ml), IgG (∼770 μg/ml), and NP-specific IgG (∼1350 ng/ml) (Supplemental Fig. 4). Similarly, cytokine-treated humanized mice were immunized twice at 2-wk interval with one of the three inactivated H5N1 virus isolates: A/Anhui/1/05 (Anhui), A/Indonesia/CDC669/06 (Idn), and A/Hong Kong/213/03 (HK). Seven days after the second immunization, sera were collected and assayed for the presence of anti-viral Abs using two methods. In the immunofluorescent assay, MDCK cells were infected with Anhui, Idn, or HK viruses, fixed, incubated with sera from the respective virus-immunized mice, and then developed with labeled Abs specific to human IgG or mouse IgG. As shown in Fig. 4A, no positive staining was detected when sera from nonimmunized GM-CSF and IL-4–treated (Ctrl) mice were used. As a positive control, when a murine anti-H5N1 mAb 2D9 (16) was used, positive signal was detected only with labeled secondary Ab specific for the mouse, but not human, IgG. In contrast, sera from all six H5N1-immunized GM-CSF and IL-4–treated mice (two for each viral isolate) reacted with infected MDCK cells when labeled secondary Ab was specific to human, but not mouse, IgG. In the neutralization assay, 100 TCID50 of virus was incubated with different dilutions of sera or 2D9 mAb and then analyzed for cytopathic effect on MDCK cells. Whereas control serum did not show any neutralizing activity, 2D9 neutralized viruses at a maximum dilution factor of 128 (starting with 100 μg/ml of Ab) (Fig. 4B). Sera from GM-CSF and IL-4–treated and immunized mice also exhibited significant neutralizing activity against respective strains of H5N1 virus isolates with maximum dilution factors of 18 for the Anhui isolate, 14 for the Idn isolate, and 22 for the HK isolate. These results show that GM-CSF and IL-4 treatment stimulates human Ab responses to a diverse set of Ags, including avian H5N1 influenza virus.
After engraftment of human HSCs into immunodeficient recipient mice, B cells are the most abundantly reconstituted human blood cell type. However, they do not make any significant IgG Ab response after immunization with specific Ag. Studies have suggested multiple defects contribute to the poor human Ab responses, including poor reconstitution of myeloid APCs, mismatch between human TCR and mouse MHC molecules, block of T and B cell maturation, and deficiency in human cytokines in humanized mice (1, 3, 4, 11, 12). In this study, we show that in humanized mice, a majority of B cells express CD5 and are blocked at the transition stage, and there is a profound deficiency of human CD209+ DCs. More importantly, we show that these defects can be partially corrected by expression of human GM-CSF and IL-4 in mice. As a result, the cytokine-treated humanized mice make significant levels of Ag-specific IgG after immunization, including neutralizing Abs specific for H5N1 avian influenza viruses.
The vast majority of human B cells in humanized mice are IgM+ (8). Consistently, we found that >85% of human CD19+ B cells are IgM+ in the spleens of normal humanized mice (Fig. 1E). We noticed that most of these B cells also express CD5, consistent with a previous report (17), whereas CD5+IgM+ B cells are only a minor fraction of the B cell population in human (18, 19). On the basis of CD5 expression, human B cells can be divided into CD5+ B1 cells and CD5− conventional B2 cells. Serum IgM is thought to be secreted primarily by CD5+ B1 cells, whereas IgG Ab responses are mediated by CD5− conventional B2 cells. The relatively high levels of serum IgM but little IgG in humanized mice is consistent with this notion and raises the possibility that the observed defect in IgG Ab response is due to the composition of B cells in humanized mice.
CD5 is also expressed on transitional human pre-naive B cells, which also express CD10 and a low level of CD40 (20). We found that the majority of human B cells (>50%) in humanized mice exhibited a phenotype of CD19+CD10+CD40lowIgM+IgD+ (Fig. 1E), indicating that the development of human B cells in humanized mice is blocked at the transitional stage. Our finding is consistent with a previous study showing accumulation of CD24int/hiCD38hi immature B cells, which composed 60% of the splenic CD19+ cells in humanized mice using the NOG mice as recipients (12). Our study extends the previous study by defining the stage at which B cell development is blocked. Furthermore, we show that maturation of B cells can be stimulated by the expression of GM-CSF and IL-4 in humanized mice, as indicated by the increased expression of IgM, CD40, CD86, and decreased expression of CD20 (Supplemental Fig. 1). Notably, cytokine treatment alone also results in a significant increase in the proportion of CD5− conventional B cells. The maturation of human B cells and development of CD5− B cells are further stimulated by immunization of cytokine-treated humanized mice. Besides further upregulation of CD40 and downregulation of CD20, HLA-DR and CD80 are upregulated and CD10 and CD19 are downregulated on human B cells in immunized mice (Fig. 3C), consistent with observation that expression of CD19 and CD20 decreases during B cell Ab responses in human (21, 22). Most profoundly, the majority of B cells become CD5− after immunization. This is even more remarkable considering that total numbers of B cells increased around 6-fold in immunized GM-CSF and IL-4–treated mice compared with mice injected with control vector. As CD10 disappears upon B cell maturation (21, 23), these results suggest that B cell development in humanized mice is likely blocked at the pre-naive B cell stage. Thus, the deficiency in B cell maturation as well as preferential development of CD5+ B1 cells are likely contributing factors to the poor IgG Ab response in humanized mice.
Correlating with the maturation of B cells, GM-CSF and IL-4 treatment stimulates a significant increase in serum IgG compared with nontreated humanized mice (Fig. 2B). After immunization, the levels of total IgM and especially total IgG are further increased, reaching a much significant level of 400–700 μg/ml, respectively. Importantly, a significant level of Ag-specific IgG is also induced to immunizing Ags TT and NP-KLH (Fig. 2C, and Supplemental Fig. 4). The level of TT-specific human IgG is at the minimum protective level in humans. As in humans, the most abundant Ig isotypes are IgG1, followed by IgG2, and then IgM and IgG4. In contrast to humanized mice that were not treated with GM-CSF and IL-4, the predominant Ig isotype is IgM. In addition, GM-CSF and IL-4–treated humanized mice were able to produce neutralizing Abs against three different isolates of H5N1 avian influenza virus. IL-4 is known to stimulate B cell Ab responses and Ig class switching. It is notable that expression of IL-4 alone is not sufficient to elevate serum IgG levels and Ag-specific IgG response, suggesting that combined GM-CSF and IL-4 treatment contributes to the improved Ab responses through additional mechanisms beyond stimulating B cell maturation.
Another major contributing factor for the improved Ab responses in GM-CSF and IL-4–treated humanized mice is the induction of CD209+ myeloid DCs and the enhanced priming of CD4 T cells. The functions of human T cells, particularly CD4+ T cells, are known to be impaired in humanized mice as they only developed very weak responses to stimulations and immunizations (1, 3, 5, 12). Specifically, it has been shown that human T cells in humanized mice are unable to produce a detectable amount of IFN-γ and IL-4 upon Ag restimulation (12). We have shown previously that GM-CSF and IL-4 treatment induces the generation of CD11c+CD209+ cells in humanized mice (11). In this study, we further demonstrated that these CD209+ DCs are functional as they upregulated expression of costimulatory molecules CD80, CD86, and HLA-DR as well as secretion of IL-12 upon stimulation with LPS. Previous reports have suggested that CD209+ DCs are critical for the initiation of T cell immune responses (15, 24), and their induction in cytokine-treated mice likely contributes to the elevated Ab responses in humanized mice through induction of CD4 T cell responses. Expression of GM-CSF and IL-4 also increased the proportion of human T cells that were CD45RA− (Supplemental Fig. 1C, 1D). As human CD45RA− T cells preferentially undergo homeostatic expansion and acquisition of memory-like phenotype in mice (25), the result suggests that expression of GM-CSF and IL-4 stimulates T cell maturation in humanized mice. Upon immunization, T cells further upregulated CD40L and HLA-DR, with a small fraction of T cells still expressing the activation marker CD25 (Fig. 3D). Furthermore, a significant fraction of human CD4 T cells responded to restimulation with TT peptide by secreting IFN-γ and IL-4 as shown by ELISPOT assay (Fig. 3E). It is worth to note that in our ELISPOT assay, total splenocytes were stimulated with TT peptide QYIKANSKFIGITE. This peptide is known to be a dominant CD4 epitope in human (26) but also can stimulate CD4+ T cell response in mice (27). Although it is not known whether the peptide was presented by human and/or mouse MHC in the ELISPOT assay, our findings clearly show that GM-CSF and IL-4 treatment stimulates induction of CD209+ DCs and effective CD4 T cell priming in humanized mice.
In summary, the poor Ag-specific IgG Ab response in humanized mice is correlated with a block of B cell development at the pre-naive B cell stage, preferential development of CD5+ B cells, and a lack of CD209+ DCs. The expression of GM-CSF and IL-4 in humanized mice can correct these defects by stimulating B cell maturation, generation of CD5− conventional B cells, and induction of CD209+ DCs. As a result, these cytokine-treated mice can make a significant Ag-specific IgG Ab response after immunization. We have now identified the specific defects in humanized mice with regard to the generation of a physiological Ab response and defined a novel approach to correct them such that this platform can be used to study Ab responses and generate novel human Abs against virulent pathogens and other clinically relevant targets.
We thank Dr. William Hwang of the Singapore Cord Blood Bank for providing cord blood, Lan Hiong Wong and Hooi Linn Loo for technical assistance, Drs. Maroun Khoury, Adam Drake, and Bettina Iliopoulou for discussion and critical review of the manuscript, and Dr. Farzad Olfat for general support.
This work was supported by the National Research Foundation Singapore through the Singapore–MIT Alliance for Research and Technology's Interdisciplinary Research Group in Infectious Disease research program. J.K.Y.C. received salary support from the National Medical Research Council, Singapore (NMRC/CSA/012/2009).
The online version of this article contains supplemental material.
Abbreviations used in this article:
A/Anhui/1/05 H5N1 virus isolate
A/Hong Kong/213/03 H5N1 virus isolate
hematopoietic stem cell
A/Indonesia/CDC669/06 H5N1 virus isolate
Madin–Darby canine kidney
4-hydroxy-3-nitrophenyl acetyl–keyhole limpet hemocyanin
virus median tissue culture infectious dose
The authors have no financial conflicts of interest.