Differential Responses of Human Regulatory T Cells (Treg) and Effector T Cells to Rapamycin

Background: The immunosuppressive drug rapamycin (RAPA) promotes the expansion of CD4+ CD25highFoxp3+ regulatory\ud T cells via mechanisms that remain unknown. Here, we studied expansion, IL-2R-c chain signaling, survival pathways and resistance to apoptosis in human Treg responding to RAPA.\ud Methodology/Principal Findings: CD4+CD25+ and CD4+CD25neg T cells were isolated from PBMC of normal controls (n = 21)\ud using AutoMACS. These T cell subsets were cultured in the presence of anti-CD3/CD28 antibodies and 1000 IU/mL IL-2 for 3 to 6 weeks. RAPA (1–100 nM) was added to half of the cultures. After harvest, the cell phenotype, signaling via the PI3K/ mTOR and STAT pathways, expression of survival proteins and Annexin V binding were determined and compared to values obtained with freshly-separated CD4+CD25high and CD4+CD25neg T cells. Suppressor function was tested in co-cultures with autologous CFSE-labeled CD4+CD25neg or CD8+CD25neg T-cell responders. The frequency and suppressor activity of Treg were increased after culture of CD4+CD25+ T cells in the presence of 1–100 nM RAPA (p,0.001). RAPA-expanded Treg were largely CD4+CD25highFoxp3+ cells and were resistant to apoptosis, while CD4+CD25neg T cells were sensitive. Only Treg upregulated anti-apoptotic and down-regulated pro-apoptotic proteins. Treg expressed higher levels of the PTEN protein than CD4+CD25neg cells. Activated Treg6RAPA preferentially phosphorylated STAT5 and STAT3 and did not utilize the PI3K/ mTOR pathway.\ud Conclusions/Significance: RAPA favors Treg expansion and survival by differentially regulating signaling, proliferation and sensitivity to apoptosis of human effector T cells and Treg after TCR/IL-2 activation

Self-Organized Criticality Theory of Autoimmunity

博士（保健学）神戸大

The Danger Is Growing! A New Paradigm for Immune System Activation and Peripheral Tolerance

Successful immune defense is a complex balancing act. In order to protect a host against invasion by harmful pathogens, an immune response must be rapid and vigorous, and must eliminate foreign invaders before their populations grow beyond control. That same immune response, however, must be selective enough to recognize and ignore commensal bacteria, environmental antigens and host tissue itself. How the immune system makes the crucial decision whether or not to attack a particular antigen has been a long-standing question central to the study of immunology. Here we show that the structure of the signaling network between regulatory T-cells and type 17 helper T-cells allows the immune system to selectively attack pathogens based on whether or not the pathogens represent a growing, and thus dangerous population. We term this mechanism for immune system activation the ‘Growth Detection Paradigm’, because it offers an entirely new explanation for immune system regulation and peripheral tolerance

Itk Derived Signals Regulate the Expression of Th-POK and Controls the Development of CD4+ T Cells

T cell development is critically dependent on both the environment and signals delivered by the T cell Receptor (TCR). The Tec family kinase Itk has been suggested to be an amplifier of signals emanating from the TCR and the loss of Itk partially affects most stages of thymopoiesis. Loss of Itk also differentially affects the development of conventional vs. non-conventional or innate memory phenotype T cells. Here, we examine whether these lineage choices are affected by a combination of TCR affinity and Itk by analyzing mice lacking Itk and carrying two TCR transgenes with differing affinities, OT-II and DO11.10. Our results show that developing thymocytes receive a gradient of signals, DO11.10>OT-II>DO11.10/Itk−/−>OT-II/Itk−/−. We also show that the development of CD4+ T cells is controlled by TCR signaling via Itk, which regulates the expression of the transcription factor, Th-POK, an enforcement factor for CD4 commitment. This results in a reduction in CD4+ T cell development, and an increase in the development of MHC class II restricted TCR transgenic CD8+ T cells that resemble non-conventional or innate memory phenotype CD8 T cells. This alteration accompanies increased expression of Runx3 and its target genes Eomesodermin, Granzyme B and Perforin in Itk null OT-II CD4+ thymocytes. All together, these data suggest that Itk plays an important role in CD4/CD8 commitment by regulating signal thresholds for the lineage commitment. Our data also suggest that the lower level of TCR signaling that occurs with a low affinity TCR in the absence of Itk can redirect some MHC class II restricted CD4+ T cell to class II-restricted CD8+ innate memory phenotype T cells

Priming of Salmonella enterica Serovar Typhi-Specific CD8+ T Cells by Suicide Dendritic Cell Cross-Presentation in Humans

T cells. effector/memory T cells. Typhi

Suppression of Glomerulonephritis in NZB/NZW Lupus Prone Mice by Adoptive Transfer of Ex Vivo Expanded Regulatory T Cells

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause characterized by expansion of autoreactive lymphocytes. Regulatory T cells (Tregs) are a component of the normal immune system and contribute to the maintenance of peripheral tolerance. Treg abnormalities have been associated with several autoimmune diseases and there is interest in the role of Tregs in SLE. We previously demonstrated that transfer of expanded CD4+CD25+CD62LHI Tregs slows the development of lupus in (NZBxNZW)F1 (B/W) mice. However in the absence of Treg specific surface antigens, cell purification remains a compromise between the breadth and purity of the population isolated. Importantly, purified populations always contain Foxp3− effector T cells (Teffs) that theoretically could exacerbate autoimmunity in the recipient. Here we explore the impact of transferring the more comprehensive, but less pure Treg subset defined by CD4+CD25+ expression on development of murine lupus. All cells were FACS sorted and expanded prior to adoptive transfer. Development of proteinuria and survival were measured. We found that exogenous expansion of CD4+CD25+ cells produced a population containing 70–85% CD4+Foxp3+Tregs. Expanded Tregs had higher CTLA-4 and Foxp3 expression, increased in vitro suppression capacity, and prolonged in vivo survival as compared to freshly isolated cells. Adoptive transfer of expanded CD4+CD25+ Tregs inhibited the onset of glomerulonephritis and prolonged survival in mice. Importantly the population of Teff contained within the adoptively transferred cells had reduced survival and proliferation capacity as compared to either co-transferred Tregs or transferred Teffs expanded in the absence of Tregs. These studies demonstrate that adoptive transfer of expanded CD4+CD25+Foxp3+Tregs has the capacity to inhibit the onset of murine lupus and that this capacity is significant despite transfer of co-cultured Teff cells. These data indicate that when co-expanded with regulatory T cells, exogenously activated Teffs from autoimmune patients may not pose a significant risk of promoting disease

Early Interferon-γ Production in Human Lymphocyte Subsets in Response to Nontyphoidal Salmonella Demonstrates Inherent Capacity in Innate Cells

Background Nontyphoidal Salmonellae frequently cause life-threatening bacteremia in sub-Saharan Africa. Young children and HIV-infected adults are particularly susceptible. High case-fatality rates and increasing antibiotic resistance require new approaches to the management of this disease. Impaired cellular immunity caused by defects in the T helper 1 pathway lead to intracellular disease with Salmonella that can be countered by IFNγ administration. This report identifies the lymphocyte subsets that produce IFNγ early in Salmonella infection. Methodology Intracellular cytokine staining was used to identify IFNγ production in blood lymphocyte subsets of ten healthy adults with antibodies to Salmonella (as evidence of immunity to Salmonella), in response to stimulation with live and heat-killed preparations of the D23580 invasive African isolate of Salmonella Typhimurium. The absolute number of IFNγ-producing cells in innate, innate-like and adaptive lymphocyte subpopulations was determined. Principal Findings Early IFNγ production was found in the innate/innate-like lymphocyte subsets: γδ-T cells, NK cells and NK-like T cells. Significantly higher percentages of such cells produced IFNγ compared to adaptive αβ-T cells (Student's t test, P<0.001 and ≤0.02 for each innate subset compared, respectively, with CD4+- and CD8+-T cells). The absolute numbers of IFNγ-producing cells showed similar differences. The proportion of IFNγ-producing γδ-T cells, but not other lymphocytes, was significantly higher when stimulated with live compared with heat-killed bacteria (P<0.0001). Conclusion/Significance Our findings indicate an inherent capacity of innate/innate-like lymphocyte subsets to produce IFNγ early in the response to Salmonella infection. This may serve to control intracellular infection and reduce the threat of extracellular spread of disease with bacteremia which becomes life-threatening in the absence of protective antibody. These innate cells may also help mitigate against the effect on IFNγ production of depletion of Salmonella-specific CD4+-T lymphocytes in HIV infection

Domain Requirements and Sequence Specificity of DNA Binding for the Forkhead Transcription Factor FOXP3

The forkhead, winged-helix transcription factor FOXP3 is preferentially expressed in T regulatory (Treg) cells and is critical for their immunosuppressive function. Mutations that abolish FOXP3 function lead to systemic autoimmunity in mice and humans. However, the manner by which FOXP3 recognizes cognate DNA elements is unclear. Here we identify an in vitro optimized DNA sequence to assess FOXP3 DNA binding by electrophoretic mobility shift assay (EMSA). The optimized sequence contains two tandem copies of a core DNA element resembling, but not identical to, the canonical forkhead (FKH) binding element. The tandem nature of this optimized FOXP3-binding oligonucleotide suggests a requirement for multimerization, and EMSA experiments confirm that both the DNA-binding FKH domain and an intact leucine-zipper domain, which mediates homo-multimerization of FOXP3, are required for DNA binding. These results establish a practical framework for understanding the molecular basis by which FOXP3 regulates gene transcription and programs Treg suppressive function

Absence of Leucine Zipper in the Natural FOXP3Δ2Δ7 Isoform Does Not Affect Dimerization but Abrogates Suppressive Capacity

BACKGROUND: Phenotype and function of regulatory T cells (Treg) largely depend on the presence of the transcription factor FOXP3. In contrast to mice, human Treg cells express isoforms of this protein. Besides the full length version (FOXP3fl), an isoform lacking the exon 2 (FOXP3Delta2) is co-expressed in comparable amounts. Recently, a third splice variant has been described that in addition to exon 2 also misses exon 7 (FOXP3Delta2Delta7). Exon 7 encodes for a leucine zipper motif commonly used as structural dimerization element. Mutations in exon 7 have been linked to IPEX, a severe autoimmune disease suggested to be caused by impaired dimerization of the FOXP3 protein. PRINCIPAL FINDINGS: This study shows that the lack of exon 7 does not affect (homo-) dimerization. Moreover, the interaction of FOXP3Delta2Delta7 to RUNX1, NFAT and NF-kB appeared to be unchanged in co-immunoprecipitation experiments and reporter gene assays, when compared to FOXP3fl and FOXP3Delta2. Nevertheless, retroviral transduction with FOXP3Delta2Delta7 failed to induce the typical Treg-associated phenotype. The expression of FOXP3-induced surface molecules such as CD25 and CTLA-4 were not enhanced in FOXP3Delta2Delta7 transduced CD4+ T cells, which also failed to exhibit any suppressive capacity. Notably, however, co-expression of FOXP3fl with FOXP3Delta2Delta7 resulted in a reduction of CD25 expression by a dominant negative effect. CONCLUSIONS: The leucine zipper of FOXP3 does not mediate dimerization or interaction with NFAT, NF-kB and RUNX1, but is indispensable for the characteristic phenotype and function in Treg cells. FOXP3Delta2Delta7 could play a role in regulating the function of the other FOXP3 isoforms and may be involved in cancer pathogenesis, as it is overexpressed by certain malignant cells

Murine Toll-Like Receptor 2 Activation Induces Type I Interferon Responses from Endolysosomal Compartments

Background: Toll-like receptors (TLRs) are among the first-line sentinels for immune detection and responsiveness to pathogens. The TLR2 subfamily of TLRs (TLR1, TLR2, TLR6) form heterodimers with each other and are thus able to recognize a broad range of components from several microbes such as yeast, Gram-positive bacteria and protozoa. Until now, TLR2 activation by bacterial ligands has long been associated with pro-inflammatory cytokines but not type I interferon responses. Methodology/Principal Findings: Using a variety of transgenic mice, here we provide in vivo and in vitro data showing that TLR2 activation does in fact induce interferon-beta and that this occurs via MyD88-IRF1 and-IRF7 pathways. Interestingly, by microscopy we demonstrate that although a cell surface receptor, TLR2 dependent induction of type I interferons occurs in endolysosomal compartments where it is translocated to upon ligand engagement. Furthermore, we could show that blocking receptor internalization or endolysosomal acidification inhibits the ability of TLR2 to trigger the induction type I interferon but not pro-inflammatory responses. Conclusion/Significance: The results indicate that TLR2 activation induces pro-inflammatory and type I interferon responses from distinct subcellular sites: the plasma membrane and endolysosomal compartments respectively. Apart from identifying and characterizing a novel pathway for induction of type I interferons, the present study offers new insights into how TLR signaling discriminates and regulates the nature of responses to be elicited against extracellular and endocytosed microbes