In Vivo Dynamical Interactions between CD4 Tregs,CD8 Tregs and CD4+CD25? Cells in Mice

Background

Regulatory T cells (Tregs) were shown to be central in maintaining immunological homeostasis and preventing the development of autoimmune diseases. Several subsets of Tregs have been identified to date; however, the dynamics of the interactions between these subsets, and their implications on their regulatory functions are yet to be elucidated.

Methodology/Principal Findings

We employed a combination of mathematical modeling and frequent in vivo measurements of several T cell subsets. Healthy BALB/c mice received a single injection of either hCDR1 - a tolerogenic peptide previously shown to induce Tregs, a control peptide or vehicle alone, and were monitored for 16 days. During this period, splenocytes from the treated mice were analyzed for the levels of CD4, CD25, CD8, CD28 and Foxp3. The collected data were then fitted to mathematical models, in order to test competing hypotheses regarding the interactions between the followed T cell subsets. In all 3 treatment groups, a significant, lasting, non-random perturbation of the immune system could be observed. Our analysis predicted the emergence of functional CD4 Tregs based on inverse oscillations of the latter and CD4⁺CD25⁻ cells. Furthermore, CD4 Tregs seemed to require a sufficiently high level of CD8 Tregs in order to become functional, while conversion was unlikely to be their major source. Our results indicated in addition that Foxp3 is not a sufficient marker for regulatory activity.

Conclusions/Significance

In this work, we unraveled the dynamics of the interplay between CD4, CD8 Tregs and effector T cells, using, for the first time, a mathematical-mechanistic perspective in the analysis of Treg kinetics. Furthermore, the results obtained from this interdisciplinary approach supported the notion that CD4 Tregs need to interact with CD8 Tregs in order to become functional. Finally, we generated predictions regarding the time-dependent function of Tregs, which can be further tested empirically in future work.     

Dynamics of CD4^＋CD25^＋ T Cells in Spleens and Mesenteric Lymph Nodes of Mice Infected with Schistosomajaponicum

CD4~ CD25~ T cells play a major role in modulating immune response,but few reports havebeen published about schistosomiasis.Here,we investigated the changes in CD4~ CD25~ T cell populations inspleens and mesenteric lymph nodes of mice infected with Schistosoma japonicum.The proportions ofCD4~ CD25~ T cells in total CD4~ T cells were analyzed by flow cytometry.CD25 and Foxp3 expression wasmeasured by real-time quantitative polymerase chain reaction.The suppressive activities of CD4~ CD25~ Tcells were detected by in vitro proliferation of splenocytes.Evidence showed that the percentage of CD4~ CD25~ T cells was the same as controls 3 weeks post-infection.At the acute stage of infection,the percentagedecreased significantly.However,at the chronic stage of infection,it rebounded to normal levels or evenhigher.The expression of the CD25 and Foxp3 showed gradual increase along with the infection progress.Invitro experiment also showed the strong suppressive effect of CD4~ CD25~ T cells,isolated during the chronicstage,on proliferation of the CD25~-splenocytes.This is the first time that the dynamics of CD4~ CD25~ T cellpopulations was demonstrated in mice infected with schistosomiasis.In conclusion,our data indicated thatCD4~ CD25~ cells might be involved in the immune modulation during S.japonicum infection,which en-hances current knowledge of the mechanisms of the immuno-downregulation and re-infection inschistosomiasis.     

CD8+ DC,but Not CD8?DC,Isolated from BCG-Infected Mice Reduces Pathological Reactions Induced by Mycobacterial Challenge Infection

Background

Tuberculosis is a mycobacterial infection causing worldwide public health problems but the available vaccine is far from ideal. Type-1 T cell immunity has been shown to be critical for host defence against tuberculosis infection, but the role of dendritic cell (DC) subsets in pathogenesis of mycobacterial infection remains unclear.

Methodology/Principal Findings

We examined the effectiveness of dendritic cell (DC) subsets in BCG-infected mice in generating immune responses beneficial for pathogen clearance and reduction of pathological reactions in the tissues following challenge infection. Our data showed that only the adoptive transfer of the subset of CD8α⁺ DC isolated from infected mice (iCD8⁺ DC) generated significant protection, demonstrated by less mycobacterial growth and pathological changes in the lung and liver tissues in iCD8⁺ DC recipients than sham-treated control mice. The adoptive transfer of the CD8α⁻DC from the infected mice (iCD8⁻ DC) not only failed to reduce bacterial growth, but enhanced inflammation characterized by diffuse heavy cellular infiltration. Notably, iCD8⁻ DC produced significantly higher levels of IL-10 than iCD8⁺ DC and promoted more Th2 cytokine responses in in vitro DC-T cell co-culture and in vivo adoptive transfer experiments.

Conclusions/Significance

The data indicate that in vivo BCG-primed CD8⁺ DC is the dominant DC subset in inducing protective immunity especially for reducing pathological reactions in infected tissues. The finding has implications for the rational improvement of the prophylactic and therapeutic approaches for controlling tuberculosis infection and related diseases.     

CD34+/M-cadherin+ Bone Marrow Progenitor Cells Promote Arteriogenesis in Ischemic Hindlimbs of ApoE?/? Mice

Background

Cell-based therapy shows promise in treating peripheral arterial disease (PAD); however, the optimal cell type and long-term efficacy are unknown. In this study, we identified a novel subpopulation of adult progenitor cells positive for CD34 and M-cadherin (CD34⁺/M-cad⁺ BMCs) in mouse and human bone marrow. We also examined the long-lasting therapeutic efficacy of mouse CD34⁺/M-cad⁺ BMCs in restoring blood flow and promoting vascularization in an atherosclerotic mouse model of PAD.

Methods and Findings

Colony-forming cell assays and flow cytometry analysis showed that CD34⁺/M-cad⁺ BMCs have hematopoietic progenitor properties. When delivered intra-arterially into the ischemic hindlimbs of ApoE^−/− mice, CD34⁺/M-cad⁺ BMCs alleviated ischemia and significantly improved blood flow compared with CD34⁺/M-cad⁻ BMCs, CD34⁻/M-cad⁺ BMCs, or unselected BMCs. Significantly more arterioles were seen in CD34⁺/M-cad⁺ cell-treated limbs than in any other treatment group 60 days after cell therapy. Furthermore, histologic assessment and morphometric analyses of hindlimbs treated with GFP⁺ CD34⁺/M-cad⁺ cells showed that injected cells incorporated into solid tissue structures at 21 days. Confocal microscopic examination of GFP⁺ CD34⁺/M-cad⁺ cell-treated ischemic legs followed by immunostaining indicated the vascular differentiation of CD34⁺/M-cad⁺ progenitor cells. A cytokine antibody array revealed that CD34⁺/M-cad⁺ cell-conditioned medium contained higher levels of cytokines in a unique pattern, including bFGF, CRG-2, EGF, Flt-3 ligand, IGF-1, SDF-1, and VEGFR-3, than did CD34⁺/M-cad⁻ cell-conditioned medium. The proangiogenic cytokines secreted by CD34⁺/M-cad⁺ cells induced oxygen- and nutrient-depleted endothelial cell sprouting significantly better than CD34⁺/M-cad⁻ cells during hypoxia.

Conclusion

CD34⁺/M-cad⁺ BMCs represent a new progenitor cell type that effectively alleviates hindlimb ischemia in ApoE^−/− mice by consistently improving blood flow and promoting arteriogenesis. Additionally, CD34⁺/M-cad⁺ BMCs contribute to microvascular remodeling by differentiating into vascular cells and releasing proangiogenic cytokines and growth factors.     

NOD-Rag2null IL-2Rγnull Mice: An Alternative to NOG Mice for Generation of Humanized Mice

We have developed NOD-Rag2^null IL-2Rγ^null (NR2G) mice similar to NOD-scidIL-2Rγ^null (NOG) mice that are known as an excellent host to generate humanized mice. To evaluate the usefulness of NR2G mice as a host for humanized mice, the engraftment rates and differentiation of human cells after human hematopoietic stem cell (HSC) transplantation were compared among NR2G, NOG, and NOD-scid mice. For this purpose, the appropriate irradiation doses to expand the niche for human stem cells in the bone marrow were first determined. As a result, 8 and 2.5 Gy in adult, and 4 and 1 Gy in newborn NR2G and NOG mice, respectively, were found to be appropriate. Next, 5 × 10⁴ human umbilical cord blood CD34⁺ cells were intravenously inoculated into irradiated adult or newborn of the immunodeficient mice. These HSC transplantation experiments demonstrated that both NR2G and NOG mice showed high engraftment rates compared with NOD-scid mice, although NOG mice showed a slightly higher engraftment rate than that for NR2G mice. However, no difference was found in the human cell populations differentiated from HSCs between NR2G and NOG mice. The HSC transplantation experiments to adults and newborns of two immunodeficient mice also revealed that the HSC transplantation into newborn mice resulted in higher engraftment rate than those into adults. These results showed that NR2G mice could be used as an alternative host to NOG mice to generate humanized mice.     

Expression of Recipient CD47 on Rat Insulinoma Cell Xenografts Prevents Macrophage-Mediated Rejection through SIRPα Inhibitory Signaling in Mice

We have previously proven that the interspecies incompatibility of CD47 is responsible for in vitro phagocytosis of xenogeneic cells by host macrophages. Utilizing an in vivo model in the present study, we investigated whether genetically engineered expression of mouse CD47 in rat insulinoma cells (INS-1E) could inhibit macrophage-mediated xenograft rejection. INS-1E cells transfected with the pRc/CMV-mouse CD47 vector (mCD47-INS-1E) induced SIRPα-tyrosine phosphorylation in mouse macrophages in vitro, whereas cells transfected with the control vector (cont-INS-1E) did not. When these cells were injected into the peritoneal cavity of streptozotocin-induced diabetic Rag2^−/−γ chain ^−/− mice, which lack T, B, and NK cells, the expression of mouse CD47 on the INS-1E cells markedly reduced the susceptibility of these cells to phagocytosis by macrophages. Moreover, these mice became normoglycemic after receiving mCD47-INS-1E, whereas the mice that received cont-INS-1E failed to achieve normoglycemia. Furthermore, injection of an anti-mouse SIRPα blocking monoclonal antibody into the mouse recipients of mCD47-INS-1E cells prevented achievement of normoglycemia. These results demonstrate that interspecies incompatibility of CD47 significantly contributes to in vivo rejection of xenogeneic cells by macrophages. Thus, genetic induction of the expression of recipient CD47 on xenogeneic donor cells could provide inhibitory signals to recipient macrophages via SIPRα; this constitutes a novel approach for preventing macrophage-mediated xenograft rejection.     

Failure of Effector Function of Human CD8+ T Cells in NOD/SCID/JAK3?/? Immunodeficient Mice Transplanted with Human CD34+ Hematopoietic Stem Cells

Humanized mice, which are generated by transplanting human CD34⁺ hematopoietic stem cells into immunodeficient mice, are expected to be useful for the research on human immune responses. It is reported that antigen-specific T cell responses occur in immunodeficient mice transplanted with both human fetal thymus/liver tissues and CD34⁺ fetal cells, but it remains unclear whether antigen-specific T cell responses occur in those transplanted with only human CD34⁺ hematopoietic stem cells (HSCs). Here we investigated the differentiation and function of human CD8⁺ T cells reconstituted in NOD/SCID/Jak3^−/− mice transplanted with human CD34⁺ HSCs (hNOK mice). Multicolor flow cytometric analysis demonstrated that human CD8⁺ T cells generated from the CD34⁺ HSCs comprised only 3 subtypes, i.e., CD27^highCD28⁺CD45RA⁺CCR7⁺, CD27⁺CD28⁺CD45RA⁻CCR7⁺, and CD27⁺CD28⁺CD45RA⁻CCR7⁻ and had 3 phenotypes for 3 lytic molecules, i.e., perforin(Per)⁻granzymeA(GraA)⁻granzymeB(GraB)⁻, Per⁻GraA⁺GraB⁻, and Per^lowGraA⁺GraB⁺. These CD8⁺ T cells failed to produce IFN-γ and to proliferate after stimulation with alloantigens. These results indicate that the antigen-specific T cell response cannot be elicited in mice transplanted with only human CD34⁺ HSCs, because the T cells fail to develop normally in such mice.     

植物类MT与植物络合肽

金属硫蛋白在哺乳动物的解毒方面民挥着重要作用，植物本身也存在类似的解毒机制。研究表明，植物存在基因编码的类ＭＴ，另外还有非基因编码的植物ＭＴ，又称植物络合肽，类ＭＴ在必需重金属离子（例如Ｚｎ＾２＋、Ｃｕ＾２＋）的代谢中挥着重要作用而植物络合肽在非必需重金属离子（例如Ｃｄ＾２＋）的解毒中起到关键作用。本文了类ＭＴ和植物络合肽之间的关系以及各自的生物合成途径、相关的结构与功能、在植物中的分布等。     

快速老化鼠脑MT1和MT3cDNA克隆及mRNA定量条件的建立

金属硫蛋白（Metellothionein,MT)基因家族的三种亚型MT1、MT2和MT3在脑组织中均有表达,它们在脑组织中具有重要的神经生理和神经调节功能,其中MT3可能是老年性痴呆（Alzheimer's disease,AD)潜在的病因学因子,本研究以快速老化鼠SAMP10脑组织总RNA为模板,通过RT-PCR扩增出MT1的编码区序列和MT33′非翻译区序列的cDNA片段。将两个片段克隆到pGEM-TEasy载体上,经测序分析确定两个cDNA片段分别为MT1编码区序列和MT33′非翻译区序列,Northern杂交结果表明,MT1和MT3mRNA在2、4、6、8四个月龄的SAMP10和SAMR1脑组织中均有表达。同时建立了MTmRNA的定量条件,为进一步研究MTmRNA增龄性变化规律及与老年性痴呆病因学的关系奠定了基础。     

Zn MT作为内源性药物的研究

金属硫蛋白(Metallo thionein,MT)分子量约6500Da,含60个氨基酸残基,其中有20个半胱氨酸,每分子含七个二价金属离子,是迄今发现对具有d~(10)结构的金属离子Zn(II),Cd(II),Cu(I)亲和力最高的一种生物活性物质。其结构的保守性、在生物界的普遍存在性以及高度的可诱导性,都预示了MT在与金属有关的细胞生物学过程中发挥着重要的作用。从发现MT     

Mice Do Not Habituate to Metabolism Cage Housing–A Three Week Study of Male BALB/c Mice

The metabolism cage is a barren, non-enriched, environment, combining a number of recognized environmental stressors. We investigated the ability of male BALB/c mice to acclimatize to this form of housing. For three weeks markers of acute and oxidative stress, as well as clinical signs of abnormality were monitored. Forced swim tests were conducted to determine whether the animals experienced behavioral despair and the serotonergic integrity was tested using an 8-OH-DPAT challenge. The metabolism cage housed mice excreted approximately tenfold higher amounts of corticosterone metabolites in feces throughout the study when compared to controls. Urinary biomarkers confirmed that these mice suffered from elevated levels of oxidative stress, and increased creatinine excretions indicated increased muscle catabolism. Changes in the core body temperature (stress-induced hyperthermia) and the fur state of the mice also indicated impaired well-being in the metabolism cage housed mice. However, monitoring body weight and feed intake was found misleading in assessing the wellbeing of mice over a longer time course, and the forced swim test was found poorly suited for studying chronic stress in mice in the present setup. In conclusion, the mice were found not to acclimatize to the metabolism cages whereby concern for animal welfare would dictate that mice should be housed in this way for as short periods as possible. The elevated degree of HPA axis activity, oxidative stress, and increased overall metabolism warrant caution when interpreting data obtained from metabolism cage housed mice, as their condition cannot be considered representative of a normal physiology.     

快速老化鼠脑MT1和MT3 cDNA克隆及mRNA定量条件的建立

金属硫蛋白 (Metallothionein ,MT)基因家族的三种亚型MT1、MT2和MT3在脑组织中均有表达 ,它们在脑组织中具有重要的神经生理和神经调节功能 ,其中MT3可能是老年性痴呆 (Alzheimer′sdisease ,AD)潜在的病因学因子。本研究以快速老化鼠SAMP10脑组织总RNA为模板 ,通过RT PCR扩增出MT1的编码区序列和MT33′非翻译区序列的cDNA片段。将两个片段克隆到pGEM○R TEasy载体上 ,经测序分析确定两个cDNA片段分别为MT1编码区序列和MT33′非翻译区序列。Northern杂交结果表明 ,MT1和MT3mRNA在 2、4、6、8四个月龄的SAMP10和SAMR1脑组织中均有表达。同时建立了MTmRNA的定量条件 ,为进一步研究MTmRNA增龄性变化规律及与老年性痴呆病因学的关系奠定了基础。     

Measurement of γHV68 Infection in Mice

γ-Herpesviruses (γ-HVs) are notable for their ability to establish latent infections of lymphoid cells¹. The narrow host range of human γ-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine γ-herpesvirus 68 (γHV68) shares extensive genetic and biological similarities with human γ-HVs and is a natural pathogen of murid rodents². As such, evaluation of γHV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during γ-HVs infection.Upon intranasal inoculation, γHV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host^3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of γHV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.Download video file.^{(81M, mov)}     

P47phox?/? Mice Are Compromised in Expansion and Activation of CD8+ T Cells and Susceptible to Trypanosoma cruzi Infection

Macrophage activation of NAD(P)H oxidase (NOX2) and reactive oxygen species (ROS) is suggested to kill Trypanosoma cruzi that causes Chagas disease. However, the role of NOX2 in generation of protective immunity and whether these mechanisms are deregulated in the event of NOX2 deficiency are not known, and examined in this study. Our data showed that C57BL/6 p47^phox−/− mice (lack NOX2 activity), as compared to wild-type (WT) mice, succumbed within 30 days post-infection (pi) to low doses of T. cruzi and exhibited inability to control tissue parasites. P47^phox−/− bone-marrow and splenic monocytes were not compromised in maturation, phagocytosis and parasite uptake capacity. The deficiency of NOX2 mediated ROS was compensated by higher level of inducible nitric oxide synthase (iNOS) expression, and nitric oxide and inflammatory cytokine (TNF-α, IFN-γ, IL-1β) release by p47^phox−/− macrophages as compared to that noted in WT controls infected by T. cruzi. Splenic activation of Th1 CD4⁺T cells and tissue infiltration of immune cells in T. cruzi infected p47^phox−/− mice were comparable to that noted in infected control mice. However, generation and activation of type 1 CD8⁺T cells was severely compromised in p47^phox−/− mice. In comparison, WT mice exhibited a robust T. cruzi-specific CD8⁺T cell response with type 1 (IFN-γ+TNF-α>IL-4+IL-10), cytolytic effector (CD8⁺CD107a⁺IFN-γ⁺) phenotype. We conclude that NOX2/ROS activity in macrophages signals the development of antigen-specific CD8⁺T cell response. In the event of NOX2 deficiency, a compromised CD8⁺T cell response is generated, leading to increased parasite burden, tissue pathogenesis and mortality in chagasic mice.     

TSC1/2 Signaling Complex Is Essential for Peripheral Na?ve CD8+ T Cell Survival and Homeostasis in Mice

The PI3K-Akt-mTOR pathway plays crucial roles in regulating both innate and adaptive immunity. However, the role of TSC1, a critical negative regulator of mTOR, in peripheral T cell homeostasis remains elusive. With T cell-specific Tsc1 conditional knockout (Tsc1 KO) mice, we found that peripheral naïve CD8⁺ T cells but not CD4⁺ T cells were severely reduced. Tsc1 KO naïve CD8⁺ T cells showed profound survival defect in an adoptive transfer model and in culture with either stimulation of IL-7 or IL-15, despite comparable CD122 and CD127 expression between control and KO CD8⁺ T cells. IL-7 stimulated phosphorylation of Akt(S473) was diminished in Tsc1 KO naïve CD8⁺T cells due to hyperactive mTOR-mediated feedback suppression on PI3K-AKT signaling. Furthermore, impaired Foxo1/Foxo3a phosphorylation and increased pro-apoptotic Bim expression in Tsc1 KO naïve CD8⁺T cells were observed upon stimulation of IL-7. Collectively, our study suggests that TSC1 plays an essential role in regulating peripheral naïve CD8⁺ T cell homeostasis, possible via an mTOR-Akt-FoxO-Bim signaling pathway.     

Evidence for Persistence of Ectromelia Virus in Inbred Mice,Recrudescence Following Immunosuppression and Transmission to Na?ve Mice

Orthopoxviruses (OPV), including variola, vaccinia, monkeypox, cowpox and ectromelia viruses cause acute infections in their hosts. With the exception of variola virus (VARV), the etiological agent of smallpox, other OPV have been reported to persist in a variety of animal species following natural or experimental infection. Despite the implications and significance for the ecology and epidemiology of diseases these viruses cause, those reports have never been thoroughly investigated. We used the mouse pathogen ectromelia virus (ECTV), the agent of mousepox and a close relative of VARV to investigate virus persistence in inbred mice. We provide evidence that ECTV causes a persistent infection in some susceptible strains of mice in which low levels of virus genomes were detected in various tissues late in infection. The bone marrow (BM) and blood appeared to be key sites of persistence. Contemporaneous with virus persistence, antiviral CD8 T cell responses were demonstrable over the entire 25-week study period, with a change in the immunodominance hierarchy evident during the first 3 weeks. Some virus-encoded host response modifiers were found to modulate virus persistence whereas host genes encoded by the NKC and MHC class I reduced the potential for persistence. When susceptible strains of mice that had apparently recovered from infection were subjected to sustained immunosuppression with cyclophosphamide (CTX), animals succumbed to mousepox with high titers of infectious virus in various organs. CTX treated index mice transmitted virus to, and caused disease in, co-housed naïve mice. The most surprising but significant finding was that immunosuppression of disease-resistant C57BL/6 mice several weeks after recovery from primary infection generated high titers of virus in multiple tissues. Resistant mice showed no evidence of a persistent infection. This is the strongest evidence that ECTV can persist in inbred mice, regardless of their resistance status.     

Resident CD11b+Ly6C? Lung Dendritic Cells Are Responsible for Allergic Airway Sensitization to House Dust Mite in Mice

Conventional dendritic cells (DCs) are considered to be the prime initiators of airway allergy. Yet, it remains unclear whether specific DC subsets are preferentially involved in allergic airway sensitization. Here, we systematically assessed the respective pro-allergic potential of individually sorted lung DC subsets isolated from house dust mite antigen (HDM)-treated donor mice, following transfer to naïve recipients. Transfer of lung CD11c⁺CD11b⁺ DCs, but not CD11c⁺CD11b⁻CD103⁺ DCs, was sufficient to prime airway allergy. The CD11c⁺CD11b⁺ DC subpopulation was composed of CD11c⁺CD11b⁺Ly6C⁺ inflammatory monocyte-derived cells, whose numbers increase in the lungs following HDM exposure, and of CD11c⁺CD11b⁺Ly6C⁻ DCs, which remain stable. Counterintuitively, only CD11c⁺CD11b⁺Ly6C⁻ DCs, and not CD11c⁺CD11b⁺Ly6C⁺ DCs, were able to convey antigen to the lymph nodes and induce adaptive T cell responses and subsequent airway allergy. Our results thus support that lung resident non-inflammatory CD11c⁺CD11b⁺Ly6C⁻ DCs are the essential inducers of allergic airway sensitization to the common aeroallergen HDM in mice.