T regulatory cells lacking CD25 are increased in MS during relapse

Dysregulation of inflammatory responses is considered to be a key element in autoreactive immune responses. T regulatory cells (Tregs) are important to maintain self-tolerance and the role of CD4⁺CD25⁺FoxP3⁺ Tregs in autoimmunity has been extensively investigated. Recently, it was shown that Tregs in systemic lupus erythematosus lacked CD25 but were biologically functional. These data warrants for further investigation of CD25^? Tregs in human autoimmunity. We analyzed relapsing–remitting multiple sclerosis (MS) patients by multicolor flow cytometry for the expression of CD3, CD4, IL2R (CD25), FoxP3, and the IL7R (CD127). Further, the level of Tregs was compared in remitting and relapsing patients and correlated with disease duration. Patients in relapse exhibited higher levels of FoxP3-positive Tregs lacking CD25 compared to healthy controls (p < 0.05), indicating that Tregs attempt to restrain immune activity during relapse. The proportion of Tregs tended to be decreased with disease duration, while CD25⁺CD4⁺ and CD25⁺CD8⁺ effector T-cell proportions were elevated and positively correlated with overall disease duration (p < 0.05). In conclusion, while MS patients in remission have normal levels of Tregs of different phenotype, relapsing patients show an increased proportion of systemic CD25^? FoxP3⁺ Tregs. With time, the proportion of Tregs decrease while effector T cells expand.     

Inhibition of Fas-mediated Apoptosis by Antigen: Implications for Lymphomagenesis

Apoptotic deletion of expanded B cell populations is essential in avoidance of autoimmune disease and immune regulation of some B cell malignancies. The role of CD4+ T cells in B cell apoptosis is evident from the high incidence of B cell tumors and autoimmunity in patients with T cell diseases such as the acquired immune deficiency syndrome (AIDS). We have previously demonstrated that in Epstein-Barr Virus (EBV) negative Burkitt's lymphoma (BL), a tumor derived from proliferating centroblasts of the germinal center, the malignant lymphocytes can be induced to express Fas (CD95) by ligation of CD40 at the B cell surface. Upon CD40 engagement, BL cells are sensitized to T-cell derived death signals provided by Fas ligand (FasL, CD95L). HBL-3 is a cell line derived from an AIDS-related BL in which the tumor IgM binds the human erythrocyte "i" antigen. To determine whether Fas-mediated apoptosis of BL cells is reduced in the context of antigen to which the tumor IgM binds, we stimulated HBL-3 cells with CD40 ligand (CD40L, CD154) in the presence and absence of human erythrocytes expressing the "i" antigen, and measured Fas-mediated apoptosis upon exposure to an agonistic anti-Fas antibody. We observed that HBL-3 cells were sensitized to Fas-mediated death by exposure to CD40L. When i+ RBCs were present, Fas-mediated apoptosis in HBL-3 cells was reduced by greater than 30%. In contrast, there was no reduction in Fas-mediated apoptosis in the presence of i &#109 (I+) RBCs. These findings demonstrate that Fas-mediated deletion of BL cells is inhibited upon surface IgM engagement by antigen for which the malignant clone has affinity.     

Separation Methods of T Cells,Natural Killer,and Dendritic Cells from Peripheral Blood of Cancer Patients using Interleukin-2 and Functional Analysis of Natural Killer Cells after Separation

We aimed to induce three different immune cell subsets from a single blood sample from cancer patients to target different biological characters of cancer cells. In the presence of 6000 IU/ml IL-2, natural killer (NK) cells adhere to plastic. By using this ability, we could separate dendritic cells, T cells, and NK cells from peripheral blood mononuclear cells. The cultured NK cells demonstrated higher nonspecific cytotoxicity against tumor cell lines than did the T cells. Furthermore, adherent NK cells demonstrated higher cytotoxicity than nonadherent NK cells, although there was no difference between adherent and nonadherent NK cells in natural cytotoxicity receptors (NKp30, NKp44, NKp46) and NKG2D expression. With these results, we confirmed that we could induce dendritic cell, T cell, and higher cytotoxic NK cells from a single blood draw, and this methodology facilitates to the use of these cells for clinical grade conditions.     

Emergence of chronic Lyme arthritis: Putting the breaks on CD28 costimulation

Lyme disease is a debilitating infection that is caused upon a bite of Borrelia burgdorferi (Bb)-infected ticks. One of the most prominent clinical manifestations is the development of chronic Lyme arthritis. Months after Bb infection, ~60% of untreated Lyme patients experience intermittent arthritic attacks that may last for years. The use of the CD28^?/? mouse in Bb infection has helped to shed light into the mechanisms that govern this inflammatory process, which seems to be tightly regulated. In this current review, the effect of immunoregulation, as well as CD28 deficiency in the development of chronic Lyme arthritis is discussed.     

Promotion and prevention of autoimmune disease by CD8+ T cells

Until recently, little was known about the importance of CD8+ T effectors in promoting and preventing autoimmune disease development. CD8+ T cells can oppose or promote autoimmune disease through activities as suppressor cells and as cytotoxic effectors. Studies in several distinct autoimmune models and data from patient samples are beginning to establish the importance of CD8+ T cells in these diseases and to define the mechanisms by which these cells influence autoimmunity. CD8+ effectors can promote disease via dysregulated secretion of inflammatory cytokines, skewed differentiation profiles and inappropriate apoptosis induction of target cells, and work to block disease by eliminating self-reactive cells and self-antigen sources, or as regulatory T cells. Defining the often major contribution of CD8+ T cells to autoimmune disease and identifying the mechanisms by which they alter the pathogenesis of disease is a rapidly expanding area of study and will add valuable information to our understanding of the kinetics, pathology and biology of autoimmune disease.     

Ex‐vivo analysis of human Natural Killer T cells demonstrates heterogeneity between tissues and within established CD4+ and CD4− subsets

Our understanding of human type 1 natural killer T (NKT) cells has been heavily dependent on studies of cells from peripheral blood. These have identified two functionally distinct subsets defined by expression of CD4, although it is widely believed that this underestimates the true number of subsets. Two recent studies supporting this view have provided more detail about diversity of the human NKT cells, but relied on analysis of NKT cells from human blood that had been expanded in vitro prior to analysis. In this study we extend those findings by assessing the heterogeneity of CD4⁺ and CD4⁻ human NKT cell subsets from peripheral blood, cord blood, thymus and spleen without prior expansion ex vivo, and identifying for the first time cytokines expressed by human NKT cells from spleen and thymus. Our comparative analysis reveals highly heterogeneous expression of surface antigens by CD4⁺ and CD4⁻ NKT cell subsets and identifies several antigens whose differential expression correlates with the cytokine response. Collectively, our findings reveal that the common classification of NKT cells into CD4⁺ and CD4⁻ subsets fails to reflect the diversity of this lineage, and that more studies are needed to establish the functional significance of the antigen expression patterns and tissue residency of human NKT cells.     

Activated γδ T cells inhibit osteoclast differentiation and resorptive activity in vitro

Extensive evidence suggests that the immune system exerts powerful effects on bone cells, particularly in chronic disease pathologies such as rheumatoid arthritis (RA). The chronic inflammatory state in RA, particularly the excessive production of T cell‐derived proinflammatory cytokines such as tumour necrosis factor (TNF)‐α and interleukin (IL)‐17, triggers bone erosions through the increased stimulation of osteoclast formation and activity. While evidence supports a role for IL‐17 and TNF‐α secreted by conventional CD4⁺ T cells in RA, recent evidence in animal models of RA have implicated γδ T cells as a major producer of pathogenic IL‐17. However, the capacity of γδ T cells to influence osteoclast formation and activity in humans has not yet been investigated widely. To address this issue we investigated the effects of γδ T cells on osteoclast differentiation and resorptive activity. We have demonstrated that anti‐CD3/CD28‐stimulated γδ T cells or CD4⁺ T cells inhibit human osteoclast formation and resorptive activity in vitro. Furthermore, we assessed cytokine production by CD3/CD28‐stimulated γδ T cells and observed a lack of IL‐17 production, with activated γδ T cells producing abundant interferon (IFN)‐γ. The neutralization of IFN‐γ markedly restored the formation of osteoclasts from precursor cells and the resorptive activity of mature osteoclasts, suggesting that IFN‐γ is the major factor responsible for the inhibitory role of activated γδ T cells on osteoclastogenesis and resorptive activity of mature osteoclasts. Our work therefore provides new insights on the interactions between γδ T cells and osteoclasts in humans.     

B6.g7 mice reconstituted with BDC2·5 non‐obese diabetic (BDC2·5NOD) stem cells do not develop autoimmune diabetes

In BDC2·5 non‐obese diabetic (BDC2·5NOD) mice, a spontaneous model of type 1 diabetes, CD4⁺ T cells express a transgene‐encoded T cell receptor (TCR) with reactivity against a pancreatic antigen, chromogranin. This leads to massive infiltration and destruction of the pancreatic islets and subsequent diabetes. When we reconstituted lethally irradiated, lymphocyte‐deficient B6.g7 (I‐A^g7+) Rag^–/– mice with BDC2·5NOD haematopoietic stem and progenitor cells (HSPC; ckit⁺Lin^–Sca‐1^hi), the recipients exhibited hyperglycaemia and succumbed to diabetes. Surprisingly, lymphocyte‐sufficient B6.g7 mice reconstituted with BDC2·5NOD HSPCs were protected from diabetes. In this study, we investigated the factors responsible for attenuation of diabetes in the B6.g7 recipients. Analysis of chimerism in the B6.g7 recipients showed that, although B cells and myeloid cells were 98% donor‐derived, the CD4⁺ T cell compartment contained ～50% host‐derived cells. These host‐derived CD4⁺ T cells were enriched for conventional regulatory T cells (T_regs) (CD25⁺forkhead box protein 3 (FoxP3)⁺] and also for host‐ derived CD4⁺CD25^–FoxP3^– T cells that express markers of suppressive function, CD73, FR4 and CD39. Although negative selection did not eliminate donor‐derived CD4⁺ T cells in the B6.g7 recipients, these cells were functionally suppressed. Thus, host‐derived CD4⁺ T cells that emerge in mice following myeloablation exhibit a regulatory phenoytpe and probably attenuate autoimmune diabetes. These cells may provide new therapeutic strategies to suppress autoimmunity.     

Effects of O-GlcNAcylation on functional mitochondrial transfer from astrocytes

Mitochondria may be transferred from cell to cell in the central nervous system and this process may help defend neurons against injury and disease. But how mitochondria maintain their functionality during the process of release into extracellular space remains unknown. Here, we report that mitochondrial protein O-GlcNAcylation is a critical process to support extracellular mitochondrial functionality. Activation of CD38-cADPR signaling in astrocytes robustly induced protein O-GlcNAcylation in mitochondria, while oxygen-glucose deprivation and reoxygenation showed transient and mild protein modification. Blocking the endoplasmic reticulum – Golgi trafficking with Brefeldin A or slc35B4 siRNA reduced O-GlcNAcylation, and resulted in the secretion of mitochondria with decreased membrane potential and mtDNA. Finally, loss-of-function studies verified that O-GlcNAc-modified mitochondria demonstrated higher levels of neuroprotection after astrocyte-to-neuron mitochondrial transfer. Collectively, these findings suggest that post-translational modification by O-GlcNAc may be required for supporting the functionality and neuroprotective properties of mitochondria released from astrocytes.