Minocycline attenuates HIV‐1 infection and suppresses chronic immune activation in humanized NOD/LtsZ‐scidIL‐2Rγnull mice

More than a quarter of a century of research has established chronic immune activation and dysfunctional T cells as central features of chronic HIV infection and subsequent immunodeficiency. Consequently, the search for a new immunomodulatory therapy that could reduce immune activation and improve T‐cell function has been increased. However, the lack of small animal models for in vivo HIV study has hampered progress. In the current study, we have investigated a model of cord blood haematopoietic progenitor cells (CB‐HPCs) ‐transplanted humanized NOD/LtsZ‐scidIL‐2Rγ^null mice in which progression of HIV infection is associated with widespread chronic immune activation and inflammation. Indeed, HIV infection in humanized NSG mice caused up‐regulation of several T‐cell immune activation markers such as CD38, HLA‐DR, CD69 and co‐receptor CCR5. T‐cell exhaustion markers PD‐1 and CTLA‐4 were found to be significantly up‐regulated on T cells. Moreover, increased plasmatic levels of lipopolysaccharide, sCD14 and interleukin‐10 were also observed in infected mice. Treatment with minocycline resulted in a significant decrease of expression of cellular and plasma immune activation markers, inhibition of HIV replication and improved T‐cell counts in HIV‐infected humanized NSG mice. The study demonstrates that minocycline could be an effective, low‐cost adjunctive treatment to regulate chronic immune activation and replication of HIV.     

Current advances in humanized mouse models

Humanized mouse models that have received human cells or tissue transplants are extremely useful in basic and applied human disease research. Highly immunodeficient mice, which do not reject xenografts and support cell and tissue differentiation and growth, are indispensable for generating additional appropriate models. Since the early 2000s, a series of immunodeficient mice appropriate for generating humanized mice has been successively developed by introducing the IL-2Rγ^null gene (e.g., NOD/SCID/γc^null and Rag2^nullγc^null mice). These strains show not only a high rate of human cell engraftment, but also generate well-differentiated multilineage human hematopoietic cells after human hematopoietic stem cell (HSC) transplantation. These humanized mice facilitate the analysis of human hematology and immunology in vivo. However, human hematopoietic cells developed from HSCs are not always phenotypically and functionally identical to those in humans. More recently, a new series of immunodeficient mice compensates for these disadvantages. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γc^null and Rag2^nullγc^null mice. In this review, we describe the current knowledge of human hematopoietic cells developed in these mice. Various human disease mouse models using these humanized mice are summarized.     

Long term human reconstitution and immune aging in NOD-Rag (−)-γ chain (−) mice

Aging of the human immune system is characterized by a gradual loss of immune function and a skewing of hematopoiesis toward the myeloid lineage, a reduction in the lymphocytic lineage, and progressive increases in senescent memory T cells at the expense of naïve T cells. Both the innate and the adaptive branches of the immune system are affected, including neutrophils, macrophages, dendritic cells and lymphocytes. Mice, the most common research model, although inexpensive, do not necessarily reflect the human immune system in terms of its interaction with infectious agents of human origin or environmental factors. This study analyzed whether a human immune system contained within the NOD-Rag (−)-γ chain (−) mouse model could realistically be used to evaluate the development and therapy of aging-related diseases. To that end lightly irradiated NOD-Rag (−)-γ chain (−) mice were injected intra-hepatically on day 1 of life with purified cord blood-derived CD34⁺ stem and progenitor cells. Multiple mice were constructed from each cord blood donor. Mice were analyzed quarterly for age-related changes in the hematopoietic and immune systems, and followed for periods up to 18–24 months post-transplant. Flow cytometric analyses were performed for hematopoietic and immune reconstitution. It was observed that NOD-Rag (−)-γ chain (−) mice could be “humanized” long-term using cord blood stem cells, and that some evidence of immune aging occurred during the life of the mice.     

CXCR3‐deficiency protects influenza‐infected CCR5‐deficient mice from mortality

Mice lacking the chemokine receptor CCR5 are susceptible to mortality from a normally non‐lethal influenza infection. Here we found that CXCR3‐deficiency rescued CCR5‐deficient (CCR5^?/?) mice from influenza‐induced mortality. The number of mononuclear phagocytes in the airways was transiently increased in CCR5^?/? mice but not in CXCR3‐CCR5 double‐deficient mice. Antigen‐specific CXCR3‐CCR5 double‐deficient CD8 effector cells were less efficient at entering the airways compared with WT or CCR5^?/? CD8 effector cells. The decrease in inflammatory cell infiltrates in CXCR3‐CCR5 double‐deficient‐infected mice correlated with a decrease in CCL2 and IFN‐γ production in the airways. Finally, CXCR3‐CCR5 double‐deficient mice that survived the primary viral challenge were protected from a lethal secondary challenge, indicating that T‐cell‐mediated protective memory was not compromised in mice lacking these chemokine receptors. In conclusion, CXCR3‐deficiency attenuated the lethal cellular immune response in CCR5^?/? influenza‐infected mice without hindering viral clearance or long‐term immunity.     

Human immune system development and survival of non‐obese diabetic (NOD)‐scid IL2rγnull (NSG) mice engrafted with human thymus and autologous haematopoietic stem cells

Immunodeficient mice bearing targeted mutations in the IL2rg gene and engrafted with human immune systems are effective tools for the study of human haematopoiesis, immunity, infectious disease and transplantation biology. The most robust human immune model is generated by implantation of human fetal thymic and liver tissues in irradiated recipients followed by intravenous injection of autologous fetal liver haematopoietic stem cells [often referred to as the BLT (bone marrow, liver, thymus) model]. To evaluate the non‐obese diabetic (NOD)‐scid IL2rγ^null (NSG)–BLT model, we have assessed various engraftment parameters and how these parameters influence the longevity of NSG–BLT mice. We observed that irradiation and subrenal capsule implantation of thymus/liver fragments was optimal for generating human immune systems. However, after 4 months, a high number of NSG–BLT mice develop a fatal graft‐versus‐host disease (GVHD)‐like syndrome, which correlates with the activation of human T cells and increased levels of human immunoglobulin (Ig). Onset of GVHD was not delayed in NSG mice lacking murine major histocompatibility complex (MHC) classes I or II and was not associated with a loss of human regulatory T cells or absence of intrathymic cells of mouse origin (mouse CD45⁺). Our findings demonstrate that NSG–BLT mice develop robust human immune systems, but that the experimental window for these mice may be limited by the development of GVHD‐like pathological changes.     

Parameters for establishing humanized mouse models to study human immunity: Analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the IL2rγ mutation

“Humanized” mouse models created by engraftment of immunodeficient mice with human hematolymphoid cells or tissues are an emerging technology with broad appeal across multiple biomedical disciplines. However, investigators wishing to utilize humanized mice with engrafted functional human immune systems are faced with a myriad of variables to consider. In this study, we analyze HSC engraftment methodologies using three immunodeficient mouse strains harboring the IL2rγ^null mutation; NOD-scid IL2rγ^null, NOD-Rag1^null IL2rγ^null, and BALB/c-Rag1^null IL2rγ^null mice. Strategies compared engraftment of human HSC derived from umbilical cord blood following intravenous injection into adult mice and intracardiac and intrahepatic injection into newborn mice. We observed that newborn recipients exhibited enhanced engraftment as compared to adult recipients. Irrespective of the protocol or age of recipient, both immunodeficient NOD strains support enhanced hematopoietic cell engraftment as compared to the BALB/c strain. Our data define key parameters for establishing humanized mouse models to study human immunity.     

Alloreactivity but Failure to Reject Human Islet Transplants by Humanized Balb/c/Rag2−/−gc−/− Mice

A human islet transplant can cure patients with type 1 diabetes. A drawback of islet transplantation is the life‐long immunosuppressive medication, often associated with severe side effects. Moreover, in spite of the immunosuppressive therapy, islets are lost in the majority of transplanted patients over time. An improved small animal model for studies on human islet allograft rejection mechanisms and the development of new measures for its prevention is clearly warranted. Here, we evaluated the potential of Balb/cRag2^?/?γc^?/? mice carrying a human‐like immune system (so‐called humanized mice) as a tool for studies on the rejection of transplanted human islets. Human T cells from Balb/cRag2^?/?γc^?/? mice, which as neonates had been transplanted with CD34⁺ human cord blood haematopoietic stem cells (HIS mice), proliferated in response to allogeneic human dendritic cells, but failed to reject a human islet allograft transplanted to the renal subcapsular space as assessed by immunohistochemistry and analysis of human serum C‐peptide levels. Histological analysis revealed that few if any T cells had migrated to the grafted tissue. These observations question the usefulness of the HIS mouse model for studies on human islet allograft rejection mechanisms and highlight the need for further improvements.     

High activation and skewed T cell differentiation are associated with low IL-17A levels in a hu-PBL-NSG-SGM3 mouse model of HIV infection

The humanized NOD/SCID/IL-2 receptor γ-chain^null (NSG) mouse model has been widely used for the study of HIV pathogenesis. Here, NSG mice with transgenic expression of human stem cell factor (SCF), granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3 (NSG-SGM3) were injected with peripheral blood leukocytes (PBL mice) from two HIV-infected (HIV⁺) patients who were under anti-retroviral therapy (ART; referred as HIV⁺ mice) or one HIV-seronegative healthy volunteer (HIV⁻). Such mice are either hu-PBL-NSG-SGM3 HIV⁺ or HIV⁻ mice, depending on the source of PBL. The kinetics of HIV replication and T cell responses following engraftment were evaluated in peripheral blood and secondary lymphoid tissues. High HIV replication and low CD4 : CD8 ratios were observed in HIV⁺ mice in the absence of anti-retroviral therapy (ART). Consistent with high activation and skewed differentiation of T cells from the HIV-infected donor, HIV⁺ mice exhibited a higher T cell co-expression of human leukocyte antigen D-related (HLA-DR) and CD38 than HIV⁻ mice, as well as a shifted differentiation to a CCR7⁻CD45RA⁺ terminal effector profile, even in the presence of ART. In addition, HIV replication and the activation/differentiation disturbances of T cells were associated with decreased plasma levels of IL-17A. Thus, this hu-PBL-NSG-SGM3 mouse model recapitulates some immune disturbances occurring in HIV-infected patients, underlying its potential use for studying pathogenic events during this infection.     

Significant involvement of nuclear factor‐κB‐inducing kinase in proper differentiation of αβ and γδ T cells

Nuclear factor‐κB‐inducing kinase (NIK) is known to play a critical role in maintaining proper immune function. This is exemplified in the spontaneous mutant mouse lacking functional NIK, alymphoplasia (aly), which is simultaneously immune‐compromised and autoimmune‐prone. To investigate the role of NIK in αβ T‐cell repertoire formation, we analysed T‐cell development in aly/aly mice bearing a transgenic T‐cell receptor (TCR). Although there were no apparent abnormalities in the mature αβ T cells of non‐transgenic aly/aly mice, the maturation efficiency of idiotype^high+ T cells in the TCR‐transgenic mice was lower in aly/aly mice compared with those found in aly/+ mice, suggesting that the mature αβ T‐cell repertoire could be altered by the absence of functional NIK. In one strain of TCR‐transgenic aly/aly mice with a negatively selecting H‐2 background, the proportion of CD8^low+ idiotype^high+ cells, which are thought to potentially represent the γδ lineage of T cells, was markedly decreased. When the γδ T cells in non‐transgenic aly/aly mice were investigated, the proportion of γδ T cells in the peripheral organs of aly/aly mice was found to be one‐half to one‐fifth of those in aly/+ mice. Analyses of bone marrow chimera mice indicated that NIK in host cells, rather than in donor cells was important for generating a normal number of peripheral γδ T cells. Collectively, these results suggest that NIK could be involved in thymic positive selection of some αβ T cells and that NIK in non‐haematopoietic cells is important for the optimal development and/or maintenance of γδ T cells.     

Reduced immunogenicity of Plasmodium falciparum gamete surface antigen (Pfs48/45) in mice after disruption of disulphide bonds – evaluating effect of interferon‐γ‐inducible lysosomal thiol reductase

Sexual stages of Plasmodium are critical for malaria transmission and stage‐specific antigens are important targets for development of malaria transmission‐blocking vaccines. Plasmodium falciparum gamete surface antigen (Pfs48/45) is important for male gamete fertility and is being pursued as a candidate vaccine antigen. Vaccine‐induced transmission‐blocking antibodies recognize reduction‐sensitive conformational epitopes in Pfs48/45. Processing and presentation of such disulphide‐bond‐constrained epitopes is critical for eliciting the desired immune responses. Mice lacking interferon‐γ‐inducible lysosomal thiol reductase (GILT), an enzyme that mediates reduction of S‐S bonds during antigen processing, were employed to investigate immunogenicity of Pfs48/45. It has been well established that the ability to reduce S‐S bonds in antigens guides effective T‐cell immune responses; however, involvement of GILT in the induction of subsequent B‐cell responses has not been explored. We hypothesized that the ability to reduce S‐S bonds in Pfs48/45 will impact the generation of T‐cell epitopes, and so influence helper T‐cell responses required for specific B‐cell responses. Non‐reduced and reduced and alkylated forms of Pfs48/45 were employed to evaluate immune responses in wild‐type and GILT knockout mice and studies revealed important differences in several immune response parameters, including differences in putative T‐cell epitope recognition, faster kinetics of waning of Pfs48/45‐specific IgG1 antibodies in knockout mice, differential patterns of interferon‐γ and interleukin‐4 secretions by splenocytes, and possible effects of GILT on induction of long‐lived plasma cells and memory B cells responsible for antigen‐recall responses. These studies emphasize the importance of antigen structural features that significantly influence the development of effective immune responses.     

T‐cell receptor activation of human CD4+ T cells shifts the innate TLR response from CXCL8hiIFN‐γnull to CXCL8loIFN‐γhi

Toll‐like receptors (TLRs) play a major part in providing innate immunity against pathogenic microorganisms. Recent studies show that these receptors are also expressed on T cells, which are the sentinels of adaptive immunity. Here, we have investigated the regulatory role of the T‐cell receptor in the functioning of these innate receptors in T cells. We show that freshly isolated human CD4⁺ T cells readily secrete the neutrophil chemoattractant CXCL8 upon activation with the TLR ligands Pam3CSK and flagellin. In contrast, TCR‐activated cells secrete considerably less CXCL8 but start producing IFN‐γ upon stimulation with TLR agonists in the absence of concomitant TCR engagement. These T cells show increased activation of p38 and JNK MAP‐kinases in response to TLR stimulation, and inhibition of p38 abrogates TLR‐induced IFN‐γ secretion. The shifting of the T‐cell innate immune response from CXCL8^hiIFN‐γ^null in freshly isolated to CXCL8^loIFN‐γ^hi in activated T cells is also observed in response to endogenous innate stimulus, IL‐1. These results suggest that the innate immune response of human CD4⁺ T cells switches from a proinflammatory to an effector type following activation of these cells through the antigen receptor.     

Human rhinoviruses induce IL‐35‐producing Treg via induction of B7‐H1 (CD274) and sialoadhesin (CD169) on DC

IL‐35 is a heterodimer of EBV‐induced gene 3 and of the p35 subunit of IL‐12, and recently identified as an inhibitory cytokine produced by natural Treg in mice, but not in humans. Here we demonstrate that DC activated by human rhinoviruses (R‐DC) induce IL‐35 production and release, as well as a suppressor function in CD4⁺ and CD8⁺ T cells derived from human peripheral blood but not in naïve T cells from cord blood. The induction of IL‐35‐producing T cells by R‐DC was FOXP3‐independent, but blocking of B7‐H1 (CD274) and sialoadhesin (CD169) on R‐DC with mAb against both receptors prevented the induction of IL‐35. Thus, the combinatorial signal delivered by R‐DC to T cells via B7‐H1 and sialoadhesin is crucial for the induction of human IL‐35⁺ Treg. These results demonstrate a novel pathway and its components for the induction of immune‐inhibitory T cells.     

High diversity of the immune repertoire in humanized NOD.SCID.γc−/− mice

The diversity of the human immune repertoire and how it relates to a functional immune response has not yet been studied in detail in humanized NOD.SCID.γc^?/? immunodeficient mice. Here, we used a multiplex PCR on genomic DNA to quantify the combinatorial diversity of all possible V–J rearrangements at the TCR‐β chain and heavy chain Ig locus. We first show that the combinatorial diversity of the TCR‐β chain generated in the thymus was well preserved in the periphery, suggesting that human T cells were not vastly activated in mice, in agreement with phenotypic studies. We then show that the combinatorial diversity in NOD.SCID.γc^?/? mice reached 100% of human reference samples for both the TCR and the heavy chain of Ig. To document the functionality of this repertoire, we show that a detectable but weak HLA‐restricted cellular immune response could be elicited in reconstituted mice after immunization with an adenoviral vector expressing HCV envelope glycoproteins. Altogether, our results suggest that humanized mice express a diversified repertoire and are able to mount antigen‐specific immune responses.     

Peripheral and Site‐Specific CD4+CD28null T Cells from Rheumatoid Arthritis Patients Show Distinct Characteristics

Proinflammatory CD4⁺CD28^null T cells are frequently found in the circulation of patients with rheumatoid arthritis (RA), but are less common in the rheumatic joint. In the present study, we sought to identify functional differences between CD4⁺CD28^null T cells from blood and synovial fluid in comparison with conventional CD28‐expressing CD4⁺ T cells. Forty‐four patients with RA, displaying a distinct CD4⁺CD28^null T cell population in blood, were recruited for this study; the methylation status of the IFNG locus was examined in isolated T cell subsets, and intracellular cytokine production (IFN‐γ, TNF, IL‐17) and chemokine receptor expression (CXCR3, CCR6 and CCR7) were assessed by flow cytometry on T cells from the two compartments. Circulating CD4⁺CD28^null T cells were significantly more hypomethylated in the CNS‐1 region of the IFNG locus than conventional CD4⁺CD28⁺ T cells and produced higher levels of both IFN‐γ and TNF after TCR cross‐linking. CD4⁺CD28^null T cells from the site of inflammation expressed significantly more CXCR3 and CCR6 compared to their counterparts in blood. While IL‐17A production could hardly be detected in CD4⁺CD28^null cells from the blood, a significant production was observed in CD4⁺CD28^null T cells from synovial fluid. CD4⁺CD28^null T cells were not only found to differ from conventional CD4⁺CD28⁺ T cells in the circulation, but we could also demonstrate that synovial CD4⁺CD28^null T cells showed additional effector functions (IL‐17 coproduction) as compared to the same subset in peripheral blood, suggesting an active role for these cells in the perpetuation of inflammation in the subset of patients having a CD28^null population.     

Natural killer cell‐mediated contact sensitivity develops rapidly and depends on interferon‐α, interferon‐γ and interleukin‐12

Natural killer (NK) cell‐mediated contact sensitivity was recently described in mice. Here, we confirm NK cell‐mediated contact sensitivity (CS) in SCID and RAG1^?/? mice but not in SCID_beige mice, which have non‐functional NK cells that lack NK cell granules. NK cell‐mediated CS was transferred by liver mononuclear cells and the DX5⁺ fraction of liver cells, confirming that NK cells mediate CS in the absence of T and B cells. Participation of NKT cells and B‐1 cells was ruled out using Jα18^?/? and JH^?/? mice, respectively. Remarkably, NK cell‐mediated CS was observed just 1 hr after immunization and was detectable as early as 30 min after challenge. Further, we examined cytokine requirements for NK cell‐mediated CS, and found that liver mononuclear cells from interleukin‐12^?/?, interferon‐γ^?/? and interferon‐α receptor^?/? donors fail to transfer NK cell‐mediated CS to naive hosts. Our studies clearly show that dinitrofluorobenzene sensitized NK cells mediate very rapid, antigen‐specific cell‐mediated immunity, with features of both innate and acquired immune responses.     

Humanized mouse as an appropriate model for accelerated global HIV research and vaccine development: current trend

Humanized mouse models currently have seen improved development and have received wide applications. Its usefulness is observed in cell and tissue transplant involving basic and applied human disease research. In this article, the development of a new generation of humanized mice was discussed as well as their relevant application in HIV disease. Furthermore, current techniques employed to overcome the initial limitations of mouse model were reviewed. Highly immunodeficient mice which support cell and tissue differentiation and do not reject xenografts are indispensable for generating additional appropriate models useful in disease study, this phenomenom deserves emphases, scientific highlight and a definitive research focus. Since the early 2000s, a series of immunodeficient mice appropriate for generating humanized mice has been successively developed by introducing the IL-2Rγ^null gene (e.g. NOD/SCID/γc^null and Rag2^nullγc^null mice) through various genomic approaches. These mice were generated by genetically introducing human cytokine genes into NOD/SCID/γc^null and Rag2^nullγc^null mouse backgrounds. The application of these techniques serves as a quick and appropriate mechanistic model for basic and therapeutic investigations of known and emerging infections.     

Deficiency of bone marrow β3‐integrin enhances non‐functional neovascularization

β3‐Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in β3‐integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on β3‐null endothelial cells. Here we transplanted β3‐null bone marrow (BM) into wild‐type (WT) mice to dissect the role of BM β3‐integrin deficiency in pathological angiogenesis. Mice transplanted with β3‐null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow β3‐integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM β3‐integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with β3‐null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with β3‐null bone marrow a significant proportion of tumour blood vessels are non‐functional when compared with tumour blood vessels in WT‐transplanted controls. Furthermore, β3‐null‐transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT‐transplanted animals. BM β3‐integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF‐induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with β3‐null bone marrow when compared with WT‐transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in β3‐null‐transplanted mice. In conclusion, although BM β3‐integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM β3‐integrin in VEGF‐induced mobilization of bone marrow‐derived cells to the peripheral circulation and for the functionality of those vessels in which BM‐derived cells become incorporated. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Humanized NOG mice as a model for tuberculosis vaccine‐induced immunity: a comparative analysis with the mouse and guinea pig models of tuberculosis

The humanized mouse model has been developed as a model to identify and characterize human immune responses to human pathogens and has been used to better identify vaccine candidates. In the current studies, the humanized mouse was used to determine the ability of a vaccine to affect the immune response to infection with Mycobacterium tuberculosis. Both human CD4⁺ and CD8⁺ T cells responded to infection in humanized mice as a result of infection. In humanized mice vaccinated with either BCG or with CpG‐C, a liposome‐based formulation containing the M. tuberculosis antigen ESAT‐6, both CD4 and CD8 T cells secreted cytokines that are known to be required for induction of protective immunity. In comparison to the C57BL/6 mouse model and Hartley guinea pig model of tuberculosis, data obtained from humanized mice complemented the data observed in the former models and provided further evidence that a vaccine can induce a human T‐cell response. Humanized mice provide a crucial pre‐clinical platform for evaluating human T‐cell immune responses in vaccine development against M. tuberculosis.