There is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an “allogenized” mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts.

An emerging field where humanized mice could prove to be useful is the study of human immune responses to allogeneic PSC transplants to assess the efficacy and safety of PSCs and guide effective immunosuppressive therapies. Here we describe the use of hSRC and hBLT humanized NSG mice to model the human immune response to allogeneic hESCs and their derivatives. We track allograft survival over time using bioluminescence imaging (BLI). In addition, we provide large transcriptome data as well as single-cell immunological analysis of human graft-infiltrating T cells and splenocytes isolated from humanized mice. Furthermore, using a similar implantation of mouse liver, thymus, and bone marrow, we developed an “allogenized” mouse model as a surrogate to assess allogeneic immunological responses to murine PSC allografts in vivo and ex vivo.

Despite enhanced engraftment of human HSCs in immunodeficient IL2rgmice, a robust human T cell-mediated immune response could not be established (). The relatively weak T cell response was hypothesized to be due to the lack of human leukocyte antigen (HLA) on the murine thymus that is necessary for the positive selection of human T cells. To address this, a new model was created by subcapsular renal implantation of human liver and thymus fragments as well as intravenous injection of autologous (human liver-derived) HSCs in sublethally irradiated immunodeficient mice and was termed the human bone marrow, liver, and thymus (hBLT) model (). The superior engraftment of human immune cells combined with positive selection of T cells in the autologous human thymus has made this the preferred model for studying human immune responses to infection ().

To model human immune responses, researchers have been studying immunodeficient mice engrafted with human immune cells and their progenitors, such as peripheral blood mononuclear cells (PBMCs) and hematopoietic stem cells (HSCs). The first description of these “humanized mouse models” dates back to 1983, when it was reported that the Prkdc(severe combined immunodeficiency, scid) mutation in CB17 mice caused B and T cell deficiency () and suggested that CB17-scid mice would be permissive for human HSC and PBMC engraftment. However, because of the high levels of host natural killer (NK) cell activity and the spontaneous generation of mouse B and T cells, this model supported only low levels of human HSC engraftment (). With the expression of human-like SIRPA in the non-obese diabetic (NOD)-scid strain, the levels of murine NK cells decreased (), resulting in heightened engraftment of human PBMCs (). However, residual activity of NK cells as well as other innate immune system functions interfered with human HSC engraftment. Moreover, NOD-scid mice developed spontaneous thymic lymphomas, resulting in a shortened lifespan. It was not until the NOD-scid mouse strain with the interleukin-2 receptor gamma chain (IL2rg)-targeted mutation (NOD.Cg-PrkdcIL2rg/Sz, NOD scid gamma [NSG]) and related NOD/shi-scid/γc(NOG) strain mice were repopulated with human HSCs (scid-repopulating cell, hSRC mouse), that superior human hematopoietic and immune cell engraftment was achieved ().

Since the first isolation of human embryonic stem cells (hESCs) () and creation of induced pluripotent stem cells (iPSCs) (), the field of regenerative medicine has been investigating the therapeutic potential of these cells for cardiac diseases (), neurological diseases (), hepatic failure (), diabetes (), and macular degeneration (). Human clinical trials in immune-privileged areas, such as the eye for macular degeneration, are ongoing for ESC derivatives () or iPSC derivatives (). However, the immunological responses toward these derivatives in less immune-privileged sites are still poorly understood (). Recently, advances have been made in tolerizing mice to accept human ESC- and iPSC-derived progenitor grafts for long-term monitoring of graft behavior (). However, to date, it is still not clear how the human immune system would respond to allogeneic human ESC or iPSC grafts. This question would need to be answered before pluripotent stem cell (PSC) therapy, including both ESCs and iPSCs, could be widely implemented in clinical practice.

To gain insights into the defective immune responses found in the allogenized mice in response to mHA-mismatched grafts, we next examined cytokine production of splenocytes from mHA-mismatched allogenized mice. Production of IFN-γ by splenocytes in response to short-term PMA and ionomycin stimulus was found to be significantly diminished compared with immunocompetent C57BL/6 mice ( Figure 7 E). Overall, the slower growth of mHA grafts in our allogenized mice shows limited effectiveness in rejecting mHA cells. However, the significant reduction of IFN-γ levels in response to PMA and ionomycin reveals an inability of the reconstituted immune system to respond appropriately to mHA grafts.

To investigate the ability of allogenized mice to reject minor histocompatibility-mismatched grafts, we next implanted mESCs with an immunological mismatch at the minor histocompatibility antigen (mHA). The mESCs from the 129S1/SvlmJ strain (H2K) were injected i.m. or i.s. into allogenized mice (donor cells from C57BL/6J mice, H2K). In contrast to immunocompetent mice (C57BL/6), allogenized mice were unable to reject mHA-mismatched mESCs at either implantation site. The rate of tumor growth in allogenized mice was slightly slower than in non-engrafted NSG mice. Nevertheless, large teratomas without signs of regression developed within 30 days in allogenized and NSG mice ( Figures 7 A–7D).

(C and D) Quantification of the BLI data from (A) and (B), respectively, normalized to the maximum radiance on day 1.

(A and B) Allogenized mice were unable to reject mHA-mismatched 129S1/SvImJ mESCs injected (A) i.m. (n = 5) or (B) i.s. (n = 5), and their BLI signal was similar to unmanipulated NSG mice (n = 4), whereas immunocompetent mice (n = 4, n = 5) rapidly rejected these grafts at both implantation sites.

Next, allograft responses in the aBM and aBLT models were examined in vivo against allogeneic miPSCs. Allogenized mice were injected with 1 × 10labeled miPSCs i.m. or i.s., and the survival of miPSC grafts was monitored longitudinally using BLI. The allogenized mice showed robust rejection of miPSC implanted i.m., similar to immunocompetent C57BL/6 mice, although rejection was delayed by ∼7 days compared with that observed in C57BL/6 mice ( Figures 6 A and 6C ). In contrast, the PSC survival kinetics in the spleens of allogenized mice were indistinguishable from immunocompetent mice ( Figures 6 B and 6D), and no differences were observed between the aBM and aBLT models with respect to their capacity to reject allografts ( Figure S6 C). Allogenized mice also developed robust immune responses that resulted in rapid rejection of hESCs, although rejection of hESCs injected i.m. or i.s. was again slightly delayed by 4–7 days compared with that of immunocompetent mice ( Figures 6 E–6H). Overall, these findings suggest that the allogeneic thymus offers an adequate microenvironment for appropriate mouse T cell development that leads to a robust allograft response. Furthermore, these data suggest that allograft responses can be modeled in reconstituted NSG mice, although there was a delay in rejection of the PSCs at some sites in the allogenized mice compared with wild-type C57BL/6 mice.

(C and D) Quantification of the BLI data from (A) and (B), respectively, normalized to the maximum radiance on day 1.

We examined the lymphoid structures in aBLT mice and compared these with the lymphoid structures in the hBLT mice, wild-type C57BL/6 mice, and unmanipulated negative control NSG mice ( Figure 5 A). H&E analysis of the spleens of hBLT mice revealed marked hematopoiesis but minimal periarteriolar lymphocyte migration ( Figures 5 C and 5E, center). Interestingly, this splenic architecture does mature over time with the development of a marginal zone by week 24 after humanization ( Figure S7 ). In dLNs, human lymphocytes were found in minimal quantities at week 12 compared with the wild-type mouse ( Figure 5 F, left and center). In contrast, the spleen of the allogenized mouse at week 12 after humanization had a very similar architecture as the wild-type C57BL/6 mouse ( Figures 5 B and 5E), with periarteriolar lymphoid sheaths but without apparent germinal center B cell areas. In addition, the lymphoid areas of allogenized mouse spleens had irregular margins, consistent with missing marginal zone-type cell development or paucity of the perilymphoid macrophage-rich collections that are part of the specialized open and closed circulation ( Figures 5 D and 5E, right). Analysis of the dLNs in the allogenized mouse revealed high levels of lymphocytes, indicating migration of the lymphocytes to peripheral lymphoid organs ( Figure 5 F, right).

(D) Allogenized mice have an architecture more similar to the wild-type C57BL/6 mouse, with periarteriolar lymphoid sheets. However, apparent germinal center B cell areas as well as marginal zones are missing, giving the periarteriolar margins an irregular shape (H&E, 10× magnification).

(C) hBLT mice show a high degree of hematopoiesis in the spleen but little periarteriolar T cell migration and an overall disorganized splenic architecture (H&E, 10× magnification).

Engraftment of donor immune cells in allogenized NSG mice was assessed by flow cytometry 12 weeks post-HSC transplantation. Donor cells were identified based on the CD45 allelic disparity between donor C57BL/6 (CD45.2) and recipient NSG (CD45.1) mice. Both the aBM and the aBLT allogenized mouse models had high levels of donor immune cell engraftment. The percentages of total leukocytes, T cells, B cells, and NK cells in peripheral blood were similar between the two models and did not differ from wild-type C57BL/6 mice ( Figure S6 B).

Our data so far have demonstrated the superior engraftment of human immune cells in the hBLT model, but the model appears to have a limited ability to mount a robust full-scale allogeneic immune response to human PSCs over time. To determine whether this is “model-dependent,” we next created allogenized mouse models by reconstituting NSG mice with allogeneic C57BL/6 mouse bone marrow (aBM mice) as well as mouse bone marrow, liver, and thymus (aBLT mice). Donor immune cells, as well as fetal liver and thymus transplanted in the abdomen, remained viable for up to one year in these models ( Figure S6 A).

To assess where in the development of the hBLT model the failure in graft rejection occurs, we next analyzed the immune cells of hBLT mice (n = 5) at different time points during the humanization process. Blood samples from five hBLT mice were drawn at week 8 (p0), 16 (p3), and 20 (p4) after humanization, and human T cells were isolated for RNA sequencing (RNA-seq). The immune profile was then compared with T cells from a healthy control human blood sample (ctrl). The immune profile of human immune cells within the hBLT model at week 8 post-humanization reveals an immune profile similar to the human control sample, but, by weeks 16 and 20, this immune profile starts to diverge and shows significant upregulation of immune profiles associated with T cell anergy and upregulation of inhibitory molecules ( Figures 4 D and 4E).

Principal-component analysis (PCA) of the fold changes of all 92 genes revealed that hBLT CD3lymphocytes isolated from the grafts grouped with the unstimulated control human T lymphocytes ( Figure 4 A). Activated human helper T cells (CD4CD45RO) and cytotoxic T cells (CD8CD45RO) isolated from hBLT spleens showed a similar grouping to their unstimulated counterparts from the healthy control ( Figures 4 B and 4C). These activated memory graft-infiltrating lymphocytes and splenocytes had an “unstimulated” phenotype upon allogeneic antigen stimulus. However, the strong correlation between graft loss and the presence of CD45ROlymphocytes seems to indicate that the graft-infiltrating lymphocytes were properly stimulated to target the grafts earlier in the response but that the immune response had subsided. Therefore, we analyzed gene expression profiles in hBLT splenocytes associated with T cell anergy and exhaustion. T cell exhaustion is described in cases of chronic infection and continuous exposure to foreign antigens and is associated with the upregulation of genes such as CTLA4, LAG3, and TIM3 (). Human activated memory helper T cells (CD4CD45RO) and activated memory cytotoxic T cells (CD8CD45RO) isolated from the spleen showed higher expression levels of these genes compared with unstimulated and stimulated PBMCs from the healthy human control ( Figure S5 C), providing initial signs for exhaustion of the human lymphocytes. A full-scale immune response cannot be mounted by these CD4CD45ROand CD8CD45ROlymphocytes, potentially explaining the inability of the hBLT model to fully reject allogeneic hESC grafts.

(E) Human T cells isolated from hBLT mice develop T cell anergy and senescence upon aging. At p0, the T cell anergy gene profile of human T cells is similar to human control T cells. However, upon aging of the hBLT mice, the anergic profile becomes increasingly present (left). A similar result was seen for the inhibitory molecules (right). Also, the engraftment of allogeneic cells at p3 did not result in an alteration from an inhibitory to a pro-inflammatory immune profile.

(D) Human immune cells isolated from hBLT mice show an impaired immune profile compared with the human control samples. Human T cells were isolated from hBLT blood at week 8 (p0), week 16 (p3), and week 20 (p4) (n = 5 mice per time point) after humanization, and the RNA expression profile was compared with T cells isolated from healthy human blood samples. At p0, the immune profile of hBLT T cells is most similar to control T cells, despite some marked increases in pro-inflammatory cytokine profiles at that time point. In contrast, other genes involved in T cell inhibition (FOXP3, TGFB1) are hardly expressed at baseline, but their expression increases over time during the aging of the mice. Interestingly, at p3, the ESC-derived ECs and HUVECs were engrafted, but this did not result in changes in the immune profile at p4.

(B and C) A similar trend was seen for activated helper T cells (CD4 + CD45RO, B) and activated cytotoxic T cells (CD8 + CD45RO, C) isolated from the spleens of hBLT mice.

Immune cells isolated from spleens, dLNs, and Matrigel plugs from hESC-EC-engrafted mice were used for mixed lymphocyte reactions to assess the effectiveness of the different lymphoid structures in mounting an immune response. First, immune cells isolated from the blood and spleen of hBLT mice were stimulated with phorbol ester (phorbol myristate acetate [PMA])/ionomycin, and their activation was compared with positive control samples of HiCK-1 cells and PBMCs isolated from human whole blood ( Figure S5 A). Even though some pro-inflammatory responses were seen in the splenocytes of hBLT mice, this was reduced compared with the positive control samples. Second, immune cells isolated from the Matrigel plugs and dLNs were incubated with hESC-EC lysate or HUVEC lysate to compare specific and non-specific immune responses ( Figure S5 B). In this experiment, neither hESC-EC nor HUVEC lysate exposure resulted in immune cell activation. Moreover, non-antigen stimulation by PMA/ionomycin also did not result in T cell activation. We next analyzed the gene expression profile of the small population of activated graft-infiltrating lymphocytes. As negative and positive controls for T cell activation, PBMCs from a healthy human donor remained unstimulated or were stimulated in vitro with PMA/ionomycin for 72 hr. After 72 hr, the PBMCs were harvested and, together with the hBLT lymphocytes, stained for murine and human CD45 markers and human CD3, CD4, CD8, CD45RA, and CD45RO. Human lymphocytes with an activated memory surface marker profile (CD4CD45RO; CD8CD45RO) were isolated using fluorescence-activated cell sorting (FACS), and their gene expression was analyzed with the Fluidigm single-cell PCR platform using a panel of 92 genes known to be involved with human Th1, Th2, and Th3 immune responses ( Table S2 ).

Taken together, our data suggest that these mice had an activated systemic immune system in the blood, but these active immune cells were not seen in similar quantities in the spleen, dLNs, or graft. Nevertheless, the limited numbers of activated graft-infiltrating T cells correlated strongly with graft loss. This revealed some functionality of the immune system in hBLT mice but also showed that they are incapable of mounting an effective alloimmune response to completely reject hESC-ECs.

It is important to note that, over the course of the 4-week experiments, the condition of the hBLT mice deteriorated, with decreased body weight, fur loss, and lessened physical activity. For three hBLT mice, this necessitated euthanization. Uncertain about the cause of this increased morbidity and mortality, we analyzed the blood, spleen, draining inguinal lymph nodes (dLNs), and PSC grafts for human immune cell populations in the remaining 12 hBLT mice at 4 weeks ( Table S1 ). Blood analyses of total CD4helper T cells showed the presence of 64.5% ± 5.2% naive (CD45RA) T cells, 11.9% ± 2.8% activated memory helper T cells (CD45RO), and 7.6% ± 3.6% effector memory (CD44) helper T cells ( Figure 3 A, left). For CD8cytotoxic T cells, the levels of naive cells decreased to 53.4% ± 6.4%, and they gained a more activated memory function with CD45ROT cell levels at 20.6% ± 2.9% and cytotoxic effector memory cells at 21.0% ± 6.3% ( Figure 3 A, right). Only a very small percentage of human CD3cells (0.4% ± 0.1%) was found in the dLNs, indicating that active human cells found in the systemic circulation were unable to traffic or localize appropriately to the dLNs ( Figure 3 B). In addition, the dLNs contained very few human B cells or antigen-presenting cells. Analysis of the spleens showed substantial engraftment of human B and T lymphocytes but few monocytes or NK cells ( Figure 3 C). The subpopulations of CD3lymphocytes consisted mainly of helper T cells and few cytotoxic T cells that were largely naive, with very few effector memory cells observed. Within the grafts, the human immune cells mainly retained a naive phenotype, with small percentages of effector memory CD4(1.3% ± 0.3%) and CD8(7.0% ± 1.3%) cells ( Figure 3 D). However, there was a strong correlation between graft loss, as indicated by the relationship between the decay in BLI signal, a reduction in the number of regulatory FoxP3T cells and naive T cells ( Figure 3 E, left and center), and an increase in the amount of activated immune cells in the graft ( Figure 3 E, right). Again, the presence of inactive and immunosuppressive immune cells in the graft environment might favor graft persistence, whereas activated immune cells facilitated graft loss.

Data are displayed as percentage (%) or number of cells (counts) within the parent population, e.g. in CD45/CD3/CD4/CD8 cells. Data are represented as mean ± SEM from one experiment (n = 12).

(E) The decrease in BLI signal (Max Radiance) in the graft was significantly correlated with a lower number of regulatory T cells (FoxP3, left) and naive T helper cells (CD45RA, center) and an increase in activated T helper cells (CD45RO, right).

(C) Analysis of the spleen reveals larger populations of B cells (CD20 + ) and T cells (CD3 + ), but subpopulations of the latter show relatively high numbers of naive (CD45RA) and few effector memory (CD44 + ) T cells.

(A and B) FACS analysis of (A) blood shows an active (CD45RO + ; CD44 + ) systemic cytotoxic T cell (CD8 + ) response but (B) little population of T cells as well as B cells and macrophages in the inguinal dLNs.

Next, we derived mature endothelial cells from the labeled hESC line (hESC-ECs) to upregulate MHC class I molecules on ESCs, which has been described during differentiation of hESCs (). Upregulation of MHC class I molecules as well as several other co-stimulatory molecules was confirmed by differentiating hESCs using the embryoid body method ( Figure S2 ). hESC-ECs (1 × 10) were then either transplanted s.c. in the back or i.m. in the gastrocnemius muscle of hBLT mice at 20 weeks after humanization, and graft survival was measured over time using BLI ( Figure 2 A). An initial decline in signal, representative of non-immune-mediated cell stress and loss during injections, was followed by stabilization of the signal and persistence of the graft for the duration of the study. Within the grafts, there appeared to be minimal immune cell infiltration ( Figures S3 D and S3E). The signal kinetics of hESC-ECs in hBLT mice were very similar or, in some cases, even improved compared with those in non-engrafted NSG mice, indicating that the reconstituted immune system might even favor graft persistence. In contrast, the signal in immunocompetent FVB mice returned to baseline within 2 weeks, indicating complete rejection of the hESC-EC grafts ( Figure 2 B). To be sure that the limited immune response was not the result of possible immune-evasive properties of hESC-ECs, we performed an additional experiment by transplanting labeled somatic human umbilical vein endothelial cells (HUVECs) into 16-week-old hBLT mice. Similar to the hESC-EC experiment, hBLT mice were unable to fully reject the HUVECs with graft persistence over a course of 3 weeks, whereas immunocompetent FVB mice rejected the cells within a week after transplantation ( Figure S4

(B) Quantification of the BLI data normalized to the maximum radiance on day 1, showing initial non-immune-mediated graft loss followed by graft persistence in the subcutaneous (top) and gastrocnemius muscle (bottom) injection sites in NSG and hBLT mice and complete rejection by the FVB mice.

(A) Representative BLI from one animal per group over the course of 4 weeks, showing persistence of hESC-ECs after s.c. and i.m. injection in NSG (center, n = 5) and hBLT (right, n = 12) mice, whereas the immunocompetent FVB mice (left, n = 4) completely rejected the grafts by day 14.

We next tested the ability of hBLT mice to reject human and mouse PSCs. Similar to the hSRC model, hBLT mice were transplanted with mouse iPSC (miPSC) grafts, generated from fibroblasts of a transgenic FVB mouse ubiquitously expressing EGFP and Luc ( Figure 1 A), or with human IFN-γ-stimulated hESC allografts ( Figure 1 B) by intra-splenic (i.s.) injection. The survival of the grafts was again monitored using BLI, with the signal normalized to the maximum radiance on day 1 of transplantation ( Figure 1 C). Despite superior engraftment of human immune cell subsets in hBLT mice compared with hSRC mice, the murine and human grafts were not rejected and underwent proliferative growth that resulted in large teratoma formation by week 4 and week 3, respectively ( Figure S3 C).

(C) Quantification of the BLI data normalized to the maximum radiance on day 1, showing intensification of the signal over time for transplanted miPSCs as well as IFN-γ-hESCs in NSG and hBLT mice, respectively, resulting in teratoma formation (left and center). In contrast, complete immune rejection of miPSCs and IFN-γ-hESCs occurred in immunocompetent C57BL/6 mice by 21 and 14 days, respectively (right).

(A and B) Representative BLI from one mouse per group, demonstrating the proliferation of miPSCs (A), and IFN-γ-hESCs (B) after i.s. injection in NSG (n = 3) and hBLT mice (n = 5) and rejection of these cells in the immunocompetent (C57BL/6) recipients (n = 4).

Having provided evidence for the inability of the hSRC model to mount strong PSC-directed immune responses, we moved to the hBLT model. hBLT mice support robust human cell engraftment of mouse lymphoid tissues and development of functional human T lymphocytes (). Indeed, total human leukocyte engraftment in hBLT mice in peripheral blood 12 weeks after humanization showed superior engraftment of B cells and CD4T cells compared with the hSRC model ( Figures S3 A and S3B).

To investigate whether low expression of major histocompatibility complex class I (MHC class I) in hESCs played a role in the failure of hSRC mice to reject these cells, hESCs were treated with interferon gamma (IFN-γ) for 24 hr prior to implantation into hSRC mice to increase expression of MHC class I and cell immunogenicity (). MHC class I, encompassing HLA A, B, and C in humans, encodes the main molecular targets of allograft rejection as well as MHC-associated incompatibilities between donor and recipient. It is also responsible for almost all acute rejection. Indeed, upregulation of MHC class I, as well as multiple other co-stimulatory molecules, was seen in hESCs upon stimulation with IFN-γ ( Figure S2 ). However, even the IFN-γ-stimulated hESCs were not rejected by hSRC mice ( Figures S1 B, S1C, S1E, and S1F). To address the possibility that the inability to reject these hESCs may be due to the hESCs modulating the immune response locally and enforcing tolerance, we transplanted hSCR mice with murine ESCs (mESCs), which should normally be rejected by human immune cells. However, these humanized mice were unable to reject murine cells as well ( Figures S1 G and S1H).

We used both the hSRC (NSG mice engrafted with HLA-A2HSCs) and hBLT (NSG mice engrafted with HLA-A2HSCs and fetal tissue) to model the allogeneic human immune responses to HLA-mismatched (HLA-A2) hESCs. The hESCs were stably transduced with a reporter construct containing the ubiquitin promoter driving firefly luciferase (Luc) and EGFP. Allogeneic HLA-A2hESCs (1 × 10) were implanted either subcutaneously (s.c.) or intramuscularly (i.m.) into hSRC mice. The hESC survival in these mice, as well as in control non-engrafted NSG and immunocompetent C57BL/6 mice, was longitudinally monitored in vivo using BLI. Both the hSRC and non-engrafted NSG mice were unable to completely reject allogeneic hESCs implanted at either injection site, whereas the immunocompetent C57BL/6 mice completely rejected the hESC grafts within 2 weeks ( Figures S1 A, S1C, S1D, and S1F).

Discussion

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Single-cell PCR analysis of the small percentage of activated (CD45RO+) lymphocytes found in the grafts, as well as splenocytes isolated from hBLT mice, showed a similar immunological phenotype to unstimulated PBMCs isolated from a healthy human donor, which contrasted sharply with PMA and ionomycin-stimulated healthy donor PBMCs. Even though the amount of CD45RO+ T cells was small in the spleens and PSC grafts of humanized mice, CD45RO+- and CD44+-expressing CD8+ lymphocytes in the blood were increased to ∼20%, providing evidence for a systemically activated immune system. Additional RNA-seq data, derived from hBLT immune blood cells at different time points during humanization, further show the development of an anergic or exhausted T cell phenotype over time after an initial activated immune profile. In vivo testing of the different immune cells from the lymphoid organs was performed after the transplant studies by re-exposing them to antigens from the transplanted cells, unencountered antigens, or a non-antigen-specific stimulus with PMA/ionomycin. However, none of the conditions described above resulted in the upregulation of pro-inflammatory cytokines and an overall inability to be activated.

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Zhao G.

Yang Y.G.

Sykes M. Human natural regulatory T cell development, suppressive function, and postthymic maturation in a humanized mouse model. We hypothesized that the education of human immune cells in the human fetal thymus failed to completely tolerize the developing human T cells to murine antigens, leading to an activated systemic immune system and a subsequent increase in morbidity and mortality in hBLT mice. This development of a wasting disease-like syndrome in hBLT mice has been described previously (), but not in all laboratories (), and is correlated with a decrease of naive human CD45RA cells in the blood, as seen in our hBLT mice. The hBLT mice in our study were screened for chimerism 12 weeks after humanization, the transplant studies were initiated 20 weeks after humanization, and the mice were euthanized 4 weeks later. Having supporting RNA-seq and single-cell PCR data that show exhaustion of the human lymphocytes, combined with data showing impaired cytokine production during that time, indicates that there is a limited time frame for modeling human immune responses in these mice.

+ T helper (Th) type 1 and CD8+ cytotoxic T cells when antigen-specific immunity develops. Under normal circumstances, NK and NKT cells show markedly increased IFN-γ secretion within hours after stimulation ( Schoenborn and Wilson, 2007 Schoenborn J.R.

Wilson C.B. Regulation of interferon-gamma during innate and adaptive immune responses. Manz, 2007 Manz M.G. Human-hemato-lymphoid-system mice: opportunities and challenges. To test the feasibility of reconstituting an NSG mouse with a functional immune system that is capable of rejecting allogeneic PSC grafts, we next created an allogenized mouse model with allogeneic murine fetal bone marrow, thymus, and liver. This allogenized mouse model and the transplanted allogeneic hematopoietic and immune system remained viable for up to a year. Functionally, our allogenized mouse was able to fully reject the human grafts and allogeneic murine grafts. However, the allogenized mouse was unable to reject the mHA-mismatched mESCs, and these cells proliferated without signs of rejection. Cytokine profile analysis of their spleens revealed significantly lower levels of IFN-γ, which has important immunostimulatory effects and is critical for effective innate and adaptive immune responses. IFN-γ is produced predominantly by NK and NK T (NKT) cells as part of the innate immune response and by CD4T helper (Th) type 1 and CD8cytotoxic T cells when antigen-specific immunity develops. Under normal circumstances, NK and NKT cells show markedly increased IFN-γ secretion within hours after stimulation (), which did not occur in our allogenized mice. The presence of a more organized lymphoid structure in the allogenized mouse compared with the humanized mouse, as well as these immune cells’ ability to be activated by allogeneic cytokines (), might provide an explanation for why the allogenized mouse was able to reject allogeneic and human grafts. However, the absence of a fully functional innate immune system, as indicated by the lower levels of IFN-γ, resulted in a delayed immune response to human and allogeneic murine grafts and an inadequate response to mHA-mismatched grafts.