Phenotypic characterization of T- and B-lymphocytes allows a dissection in functional different subsets. Here, we questioned whether changes in peripheral lymphocyte subset distribution reflect specific clinical and histopathological entities after renal transplantation. 65 Renal transplant recipients with either histologically proven (sub)clinical acute rejection, chronic allograft dysfunction or without abnormalities were studied for their peripheral lymphocyte subset composition and compared to 15 healthy control individuals. Naïve, memory and effector CD8+ T cell counts were measured by staining for CD27, CD28 and CD45R0/RA. In addition, we studied the CD25⁺CD4⁺ T cell population for their composition regarding regulatory FOXP3⁺CD45R0⁺CD127- cells and activated CD45R0⁺CD127⁺ cells. Naïve, non-switched and switched memory B cells were defined by staining for IgD and CD27. We found a severe decrease in circulating effector-type CD8⁺ T cells in recipients with chronic allograft dysfunction at 5 years after transplantation. Percentages of circulating CD25⁺CD127^low CD4⁺ regulatory T cells after transplantation were reduced, but we could not detect any change in percentage of CD127⁺CD45R0⁺ CD4⁺ activated T cells in patients at any time or condition after renal transplantation. Regardless of clinical events, all renal transplant recipients showed decreased total B cell counts and a more differentiated circulating B cell pool than healthy individuals.

The changes in lymphocyte subset distribution probably reflect chronic antigenic stimulation that occurs in these transplant recipients. To determine the usefulness of lymphocyte subset-typing in clinical practice, large cohort studies are necessary.

© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd, Immunology

Mesenchymal stem cells (MSCs) are multipotent cells that are being clinically explored as regenerative therapeutics. Cultured MSCs secrete various modulatory factors, which contribute to the immunosuppressive effects of transplanted MSCs as a therapy. Although the in vitro phenotype of MSCs has been well characterized, identification of MSCs in vivo is difficult due to the lack of specific markers. Current advances in murine MSCs research provide valuable tools for studying the localization and function of MSCs in vivo. Recent findings suggest that MSCs exert diverse functions depending on tissue context and physiological conditions. This review focuses on bone marrow MSCs and their roles in hematopoiesis and immune responses.

© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd, Immunology

Natural killer (NK) group 2D (NKG2D) is a key activating receptor expressed on NK cells, whose interaction with ligands on target cells plays an important role in tumorigenesis. However, the effect of histamine on NKG2D ligands on tumor cells is unclear. Here we showed that human monocytic leukemia THP-1 cells constitutively express MHC class I-related chain A (MICA) and UL16-binding protein 1 on their surface, and incubation with histamine reduced the expression in a dose and time-dependent manner as assessed by flow cytometry. Interferon-γ augmented the surface expression of the NKG2D ligands, and this augmentation was significantly attenuated by histamine. The histamine H1 receptor (H1R) agonist 2-pyridylethylamine and H2R agonist dimaprit down-regulated the expression of NKG2D ligands, and activation of H1R and H2R signaling by A23187 and forskoline, respectively, had the same effect, indicating that the histamine-induced down-regulation of NKG2D ligands is mediated by H1R and H2R. Quantitative RT-PCR showed that mRNA levels of the NKG2D ligands and relevant microRNAs were not significantly changed by histamine. Histamine down-regulated the surface expression of endoplasmic reticulum protein 5, and inhibition of matrix metalloproteinases did not impair this down-regulation, indicating that proteolytic shedding is not involved. Instead, pharmacological inhibition of protein transport and proteasome abrogated it, and histamine enhanced ubiquitination of MICA. Furthermore, histamine treatment significantly reduced susceptibility to NK cell-mediated cytotoxicity. These results suggest that histamine down-regulates NKG2D ligands through the activation of an H1R- and H2R-mediated ubiquitin-proteasome pathway and consequently reduces susceptibility to NK cells.

© 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd, Immunology

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of cells that negatively regulate the immune response during tumor progression, inflammation and infection. Only limited data is available on human MDSC due to lack of specific markers. We have identified members of the S100 protein family - S100A8, S100A9 and S100A12 specifically expressed in CD14⁺HLA-DR^-/low MDSC. S100A9 staining in combination with anti-CD14 could be used to identify MDSC in whole blood from patients with colon cancer. An increase in the population of CD14⁺S100A9^high MDSC was observed in the peripheral blood from colon cancer patients in comparison to healthy controls. Finally, nitric oxide synthase (NOS2) expression, a hallmark of MDSC, was induced in CD14⁺S100A9^high upon LPS/IFN-γ stimulation. We propose S100 proteins as useful markers for the analysis and further characterization of human myeloid derived suppressor cells.

Published 2012. This article is a U.S. Government work and is in the public domain in the USA

B cell activation is triggered by the binding of antigen to the B cell receptor (BCR). The early molecular events triggered by BCR binding of ligand have been well characterized both biochemically and using optical microscopy techniques to visualise B cell activation as it happens. However we understand much less about the BCR prior to activation. Thus, this review will address recent advances in our view of the structure, organisation, and dynamics of the resting, unstimulated BCR. These parameters have important implications for our understanding of the initiation of B cell activation and will be discussed in the context of current models for BCR activation. These models include the conformation induced oligomerisation model, in which binding of antigen to monomeric BCRs induces a pulling or twisting force causing conformational unmasking of a clustering interface in the Cμ4 domain. Conversely, the dissociation activation model proposes that BCRs exist in auto-inhibitory oligomers on the resting B cell surface and binding of antigen promotes the dissociation of the BCR oligomer exposing phosphorylation residues within Igα/Igβ. Finally, the collision coupling model suggests that BCRs are segregated from activating co-receptors or kinases and activation is associated with changes in BCR mobility on the cell surface which allows for the functional interaction of these elements.

Natural killer T (NKT) cells are a heterogeneous population of lymphocytes, which recognize antigens presented by CD1d and have attracted attention due to their potential role linking innate and adaptive immune responses. Peripheral NKT cells display a memory-activated phenotype and can rapidly secrete large amounts of pro-inflammatory cytokines upon antigenic activation. In this study, we evaluated NKT cells in the context of patients co-infected with HIV-1 and Mycobacterium leprae. The volunteers were enrolled into four groups: 22 healthy controls, 23 HIV-1-infected patients, 20 patients with leprosy and 17 patients with leprosy and HIV-1-infection. Flow cytometry and ELISPOT assays were performed on Peripheral Blood Mononuclear Cells (PBMC). We demonstrated that HIV-1 and Mycobacterium leprae co-infected patients have significantly lower in NKT cell frequencies (median 0.022%, IQR:0.007 – 0.051) in the peripheral blood when compared to healthy subjects (median 0.077%, IQR:0.032 – 0.405, p<0.01) or HIV-1 mono-infected patients (median 0.072%, IQR:0.030 – 0.160, p<0.05). Also, more NKT cells from co-infected patients secreted IFN-γ after stimulation with DimerX, when compared to leprosy mono-infected patients (p=0.05). These results suggest that NKT cells are decreased in frequency in HIV-1 and Mycobacterium leprae co-infected patients compared to HIV-1 mono-infected subjects alone, but are at a more activated state. Innate immunity in human subjects is strongly influenced by their spectrum of chronic infections, and in HIV-1 infected subjects, a concurrent mycobacterial infection likely hyper-activates and lowers circulating NKT cell numbers.

Wild-type (WT) and CD1d^−/− (NKT-less) mice were treated with zymosan A to induce granuloma formation in the liver. Increased granuloma formation was seen in NKT-less mice on Days 7 and 14 after administration. WT mice showed limited granuloma formation, and zymosan A eventually induced NKT cell accumulation as identified by their surface marker (e.g. CD1d-tetramer). Zymosan A augmented the expression of TLR2 on the cell surface of both macrophages and NKT cells. One possible reason for accelerated granuloma formation in NKT-less mice was increased production of IFNγ; a theory confirmed using IFNγ^−/− mice. Also, zymosan A increased IL-10 production in WT mice, which suppresses IFNγ production. Taken together, these results suggest that NKT cells in the liver have the potential to suppress zymosan A-mediated granuloma formation.

The cellular innate immune response is essential for recognizing and defending against viral infection. RIG-I and VISA mediated immune signaling is critically involved in RNA virus induced innate immune responses. Here we demonstrate that the complement C1qA interacts with different RIG-I pathway components and enhances RIG-I-VISA mediated signaling pathway as well as TBK1 mediated activation of IFN-β promoter. Our data show that ovexpression of C1qA upregulates RIG-I mediated activation of ISRE and NF-κB reporters and IFN-β transcription, but not IRF-3 and IKK-β mediated activation of ISRE and NF-κB promoter. In addition, C1qA can counteract with the function of C1q receptor gC1qR in RIG-I mediated signaling. Our results reveal the important role of complement C1qA in innate immune response.

The presence and persistence of alloantigen is necessary for graft specific T cell mediated immunity. However, specificity comprises only a single facet of an extremely complex process. Evidence is accruing to suggest that immunogenicity could be manipulated by endogenous ligands released during tissue injury. Stress molecules are significantly upregulated following transplantation and stimulate conserved receptors on a range of leukocytes, including dendritic cells (DC). DC are essential for costimulation and the induction of adaptive immunity. Stress signals can act as an adjuvant leading to DC maturation and activation. DC stimulated by endogens exhibit enhanced alloantigen presentation, costimulation and production of proinflammatory cytokines, such as IL-1beta and IL-18. Inflammasomes have a major role in IL-1beta/IL-18 production and secretion, and can be stimulated by endogens. Importantly, the polarisation toward inflammatory T helper (Th) 17 as opposed to regulatory T (Treg) cells is dependent upon, amongst other factors, IL-1beta. This highlights an important differentiation pathway that may be influenced by endogenous signals. Minimising graft damage and stress expression should hypothetically be advantageous, and we feel that this area warrants further research, and may provide novel treatment modalities with potential clinical benefit.

The Toll-like receptors 3, 7, 8 and 9 stimulate innate immune responses upon recognizing pathogen-derived nucleic acids. TLR3 is located on the cell surface and in cellular endosomes and recognizes double-stranded viral RNA or the synthetic mimic poly rI:rC. Recently, unformulated siRNA has been reported as ligand for surface expressed murine TLR3. Blockage of TLR3 is achieved by single-stranded DNA. We confirm and expand the observation that poly rI:rC-mediated TLR3 immune activation is blocked in a sequence-, length-, backbone- and CpG-dependent manner. However, human TLR3 is not activated by siRNA, which may be due to differences in the amino acid composition of TLR3 loop 1 of mice and humans. Although CD14 was previously described as a co-receptor for murine TLR3 and other nucleic acid-recognizing TLRs, human CD14 acts only as co-receptor to human TLR9, but not TLR3, TLR7 or TLR8. We show that CD14 up-regulates the TLR9 immune response of A, B and C-class ODN but down-regulates the phosphoro-diester version of B-class ODN.

Class switching and plasma cell differentiation occur at high level within all mucosa associated lymphoid tissues. The different classes of membrane Ig heavy chains (HC) are associated with the Igα/Igβ heterodimer within the B cell receptor (BCR). Whether BCR isotypes convey specific signals adapted to the corresponding differentiation stages remains debated but IgG and IgA membrane have been suggested to promote plasma cell differentiation. We investigated the impact of blocking expression of the IgA-class BCR through a “αΔtail” targeted mutation, deleting the Cα Ig gene membrane exon. This allowed to evaluate to what extent class switching and plasma cell differentiation can be concurrent processes, allowing some αΔtail^+/+ B cells with an IgM BCR to directly differentiate into IgA plasma cells and yield serum secreted IgA in spite of the absence of membrane IgA+ B lymphocytes. By contrast in secretions, secretory IgA was very low, indicating that J-chain positive plasma cells producing sIgA overwhelmingly differentiate from previously class-switched mIgA+ memory B cells. In addition and while MALT are a major site for plasma cell accumulation, αΔtail^+/+ mice showed that the gut B lineage homeostasis is not polarized toward plasma cell differentiation through a specific influence of the mIgA BCR.

The pattern-recognition receptor (PRR) family includes Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), C-type lectin receptors (CLRs) and the receptor for advanced glycation end products (RAGE). They recognize various microbial signatures or host-derived danger signals and trigger an immune response. Eosinophils are multifunctional leukocytes involved in the pathogenesis of several inflammatory processes, including parasitic helminth infection, allergic diseases, tissue injury and tumor immunity. Human eosinophils express several PRRs, including TLR1-5, TLR7, TLR9, NOD1, NOD2, Dectin-1 and RAGE. Receptor stimulation induces survival, oxidative burst, activation of the adhesion system and release of cytokines [interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and granulocyte macrophage colony-stimulating factor (GM-CSF)], chemokines [IL-8 and growth-related oncogene (GRO)-α] and cytotoxic granule proteins [eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), eosinophil peroxidase (EPO) and major basic protein (MBP)]. It is also evident that eosinophils play an immunomodulatory role by interacting with surrounding cells. The presence of a broad range of PRRs in eosinophils indicates that they not only are involved in the defense against parasitic helminths, but also against bacteria, viruses and fungi. From a clinical perspective, eosinophilic PRRs seem to be involved in both allergic and malignant diseases by causing exacerbations and affecting tumor growth, respectively.

Dendritic cells (DCs) are known as antigen presenting cells and play a central role in both innate and acquired immunity. Peripheral blood monocytes give rise to resident and recruited DCs in lymph nodes and non-lymphoid tissues. The ligands of nuclear hormone receptors can modulate DC differentiation and thus influence various biological functions of DCs. The role of bile acids (BAs) as signaling molecules has recently become apparent, but the functional role of BAs in DC differentiation has not yet been elucidated. We show that DCs derived from human peripheral blood monocytes cultured with a BA produce lower levels of interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α) in response to stimulation with commensal bacterial antigens.

Stimulation through the nuclear receptor farnesoid X (FXR) did not affect the differentiation of DCs. However, DCs differentiated with the specific agonist for TGR5, a transmembrane BA receptor, showed an IL-12 hypo-producing phenotype. Expression of TGR5 could only be identified in monocytes and was rapidly downregulated during monocyte differentiation to DCs. Stimulation with 8-Bromoadenosine-cyclic AMP (8-Br-cAMP), which acts downstream of TGR5 signaling, also promoted differentiation into IL-12 hypo-producing DCs. These results indicate that BAs induce the differentiation of IL-12 hypo-producing DCs from monocytes via the TGR5-cAMP pathway.

Oxidized low-density lipoprotein (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to formation of lipid-laden macrophages (foam cells). Fcγ receptors mediate uptake of oxLDL-IC, whereas scavenger receptors internalize oxLDL. We have previously reported that oxLDL-IC but not free oxLDL activate macrophages and prolong their survival. Sphingomyelin is a major constituent of cell membranes and lipoprotein particles and acid sphingomyelinase (ASMase) hydrolyzes sphingomyelin to generate the bioactive lipid ceramide. ASMase exists in two forms: lysosomal (L-ASMase) and secretory (S-ASMase). In this study we examined whether oxLDL and oxLDL-IC regulate ASMase differently, and whether ASMase mediates monocyte/macrophage activation and cytokine release. oxLDL-IC but not oxLDL induced early and consistent release of catalytically active S-ASMase. oxLDL-IC also consistently stimulated L-ASMase activity, whereas oxLDL induced a rapid transient increase in L-ASMase activity before it steadily declined below baseline. Prolonged exposure to oxLDL increased L-ASMase activity; however, activity remained significantly lower than that induced by oxLDL-IC. Further studies were aimed at defining the function of the activated ASMase. In response to oxLDL-IC, heat shock protein 70B’ (HSP70B’) was up-regulated and localized with redistributed ASMase in the endosomal compartment outside the lysosome. Treatment with oxLDL-IC induced the formation and release of HSP70- and IL-1β-containing exosomes via an ASMase-dependent mechanism. Taken together, the results suggest that oxLDL and oxLDL-IC differentially regulate ASMase activity, and the pro-inflammatory responses to oxLDL-IC are mediated by prolonged activation of ASMase. These findings may contribute to increased understanding of mechanisms mediating macrophage involvement in atherosclerosis.

Systemic lupus erythematosus (SLE) is a multisystem chronic inflammatory disease affecting many organs. The deposition in kidney tissue of immune complexes and their interaction with macrophages is thought to trigger the inflammatory response leading to glomerulonephritis. It has been demonstrated that inhibition of this interaction in murine models can alleviate the disease. Six synthetic peptides were derived from the membrane-proximal extracellular domain (EC2) of huFcγRII. Of these, one peptide, huRII6, was shown to be a potent competitive inhibitor of IgG binding to recombinant FcγRII in vitro. In order to examine the possible therapeutic impact of huRII6 in vivo, this peptide, or a control was given by subcutaneous injection to female MRL/lpr mice from weeks 7 to 36, resulted in an enhanced survival rate compared to control-treated animals and a reduction of proteinuria. Histopathological examination (HE) of the kidneys showed a reduction in immune complexes (ICs) deposition and preservation of structure. Such a functional peptide should prove useful for examining the role of IgG-FcγR interactions in experimental models of disease and may provide for the development of FcR-targeting drugs to treat autoimmune disorders.

Integrins not only mediate cell-cell and cell-extracellular matrix adhesion, but also affect the multitude of signal transduction cascades in control of cell survival, proliferation, differentiation and organ development. Mutations in integrins or the major effectors of integrin signaling pathways cause defective organ development, immunodeficiency, cancer or autoimmune disease. Understanding of the signaling events that drive integrin activation and signaling is therefore crucial to uncover the molecule mechanisms of these diseases. This review discusses the key signaling complexes regulating integrin activation and function in both ‘inside-out’ and ‘outside-in’ pathways in T lymphocytes, including kinases, SLP-76, VAV1, ADAP, SKAP-55, RapL, RIAM, Rap1, Talin and Kindlin.

The immune system is intricately regulated allowing potent effectors to expand and become rapidly mobilized after infection, while simultaneously silencing potentially detrimental responses that averts immune-mediated damage to host tissues. This relies in large part on the delicate interplay between immune suppressive regulatory CD4⁺ T cells (Tregs) and immune effectors that without active suppression by Tregs cause systemic and organ-specific autoimmunity. Although these beneficial roles have been classically described to be counter-balanced by impaired host defense against infection, newfound protective roles for Tregs against specific viral pathogens (e.g. Herpes simplex-2, Lymphocytic choriomeningitis, West Nile virus) have been uncovered using transgenic mice that allow in vivo Treg-ablation based on Foxp3-expression. In turn, Foxp3⁺ Tregs also provide protection against some parasitic (Plasmodium sp., Toxoplasma gondii) and fungal (Candida albicans) pathogens. By contrast for bacterial and mycobacterial infections (e.g. Listeria monocytogenes, Salmonella enterica, Mycobacterium tuberculosis), experimental manipulation of Foxp3⁺ cells continue to indicate detrimental roles for Tregs in host defense. This discordance is likely related to functional plasticity in Treg suppression that shifts discordantly following infection with different types of pathogens. Furthermore, the efficiency whereby Tregs silence immune activation coupled with the plasticity in Foxp3⁺ cell activity suggest overriding Treg-mediated suppression represents a prerequisite “signal zero” that together with other stimulation signals (T cell receptor [signal 1], co-stimulation [signal 2], inflammatory cytokines [signal 3]) are essential for T cell activation in vivo. Herein, the importance of Foxp3⁺ Tregs in host defense against infection, and the significance of infection-induced shifts Treg suppression are summarized.

Highly differentiated CD8⁺CD28^-CD27^- T cells have short telomeres, defective telomerase activity and reduced capacity for proliferation. In addition, these cells express increased levels of inhibitory receptors and display defective Akt(ser⁴⁷³) phosphorylation following activation. It is not known whether signalling via PD-1 contributes to any of the attenuated differentiation-related functional changes in CD8⁺ T cells. To address this we blocked PD-1 signalling during TCR activation using antibodies against PDL1 and PDL2. This resulted in a significant enhancement of Akt(ser⁴⁷³) phosphorylation and TCR-induced proliferative activity of highly differentiated CD8⁺CD28^-CD27^- T cells. In contrast, the reduced telomerase activity in these cells was not altered by blockade of PDL-1/2. We also demonstrate that PD-1 signaling can inhibit the proliferative response in primary human CD8⁺ T cells from both young and older humans. This data collectively highlights that certain some but not all functional changes that arise during progressive T cell differentiation and during ageing are maintained actively by inhibitory receptor signaling.

Understanding the thymic processes that support the generation of functionally competent and self-tolerant lymphocytes requires dissection of the T cell receptor (TCR) response to ligands of different affinities. In spatially segregated regions of the thymus, with unique expression of proteases and cytokines, TCR affinity guides a number of cell fate decisions. Yet affinity alone does not explain the selection paradox. Increasing evidence suggests that the ‘altered peptide’ model of the 1980′s together with the affinity model might best explain how the thymus supports conventional and regulatory T cell development. Development of new tools to study the strength of TCR signals perceived by T cells, novel Treg transgenic mice, and tetramer enrichment strategies have provided an insight into the nature of TCR signals perceived during thymocyte development. These topics are discussed and support for the prevailing hypotheses are presented.

Effective immune responses require antigen uptake by APC, followed by controlled endocytic proteolysis resulting in the generation of antigen-derived peptide fragments that associate with intracellular MHC class II molecules. Resultant peptide/MHCII complexes then move to the APC surface where they activate CD4⁺ T cells. DC, macrophages and B cells act as efficient APC. In many settings, including the T_H1-dependent, proteoglycan-induced arthritis (PGIA) model of rheumatoid arthritis (RA), accumulating evidence demonstrates that antigen presentation by B cells is required for optimal CD4⁺ T cell activation. The reasons behind this however, remain unclear. In this study we have compared the activation of CD4⁺ T cells specific for the proteoglycan aggrecan following antigen presentation by DC, macrophages and B cells. We show that aggrecan-specific B cells are equally efficient APC as DC and macrophages and use similar intracellular antigen processing pathways. Importantly, we also show that antigen presentation by aggrecan-specific B cells to TCR transgenic CD4⁺ T cells results in enhanced CD4⁺ T cell IFN-γ production and T_H1 effector sub-set differentiation compared to that seen with DC. We conclude that preferential CD4⁺ T_H1 differentiation may define the requirement for B cell APC function in both PGIA and RA.

Deficiency in Daf1, a complement regulatory protein, has been shown to exacerbate development of various autoimmune diseases and recent studies have suggested this may be explained by Daf1 acting to limit T cell hyper-responsiveness. It has been suggested that the absence of Daf1 aggravates autoimmune disease in a complement dependent manner, but others have shown that activation of T cells in the absence of Daf1 can be complement independent. However, the relationship between Daf1, complement components, lymphocyte activation, cytokine expression and antibody production remains to be determined in mice that are not Daf1 deficient. We have recently demonstrated, in murine mercury-induced autoimmunity (mHgIA), that an accumulation of CD44^high Daf^low CD4⁺ T cells is associated with the development of autoimmunity. In this study we observed that complement depletion does not affect the accumulation of activated CD4⁺ T cells, elevation of splenic IL-4 expression and autoantibody production in mHgIA. In addition, neither the accumulation of CD44^high Daf^low CD4⁺ T cells nor the downregulation of Daf1 expression on CD4⁺ T cells were influenced by a lack of complement. In conclusion, these studies show that initiating events in xenobiotic-induced autoimmunity, including lymphocyte activation, cytokine expression and autoantibody production, are not dependent on complement.

Diabetes mellitus is a predisposing factor of melioidosis contributing to higher mortality rates in diabetics infected with Burkholderia pseudomallei. To investigate how diabetes alters the inflammatory response, we established a streptozotocin (STZ)-induced diabetic murine acute-phase melioidosis model. Viable B. pseudomallei cells were consistently detected in the blood, liver and spleen during the 42 hr course of infection but the hyperglycemic environment did not increase the bacterial burden. However after 24 hours, granulocyte counts increased in response to infection, whilst blood glucose concentrations decreased over the course of infection. A genome wide expression analysis of the STZ-diabetic murine acute melioidosis liver identified ∼1000 genes whose expression was altered in the STZ-diabetic mice. The STZ-diabetic host transcriptional response was compared with the normoglycemic host transcriptional response recently reported by our group. The microarray data suggests that presence of elevated glucose levels impairs the host innate immune system by delaying the identification and recognition of B. pseudomallei surface structures. Consequently, the host is unable to activate the appropriate innate immune response over time, which may explain the increased susceptibility to melioidosis in the STZ-diabetic host. Nevertheless, a general “alarm signal” of infection as well as defence programs are still triggered by the STZ-diabetic host, however, only after 24 hr post infection. In summary, this study demonstrates that in the face of a B. pseudomallei acute infection, poor glycemic control impaired innate responses during the early stages of B. pseudomallei infection, contributing to STZ-induced diabetics increased susceptibility to this fatal disease.

Experimental models have shown that lipoproteins from Mycobacterium tuberculosis (Mtb) induce apoptosis via TLR2 in the THP-1 cell line and in monocyte-derived macrophages from healthy volunteers. We found an increased percentage of circulating monocytes in tuberculosis (TB) patients in comparison to healthy controls. TB patients showed a higher TLR2 and TLR4 expression density on monocytes, and a higher proportion of TLR2⁺ monocytes, as well as increased serum TNF-alpha level. In culture, monocytes from TB patients were more susceptible to undergo death than monocytes from healthy controls. Moreover, death susceptible monocytes were positive to both TLR2 and TLR4 at the start of culture. Freshly obtained monocytes from TB patients exhibited cleaved caspase 9 and denaturalized cytochrome-C. Regarding caspase 8, ASK1, and phospho-p38 MAPK levels, no difference was observed between samples from TB patients and healthy controls. The culture filtrate antigen extract (CFAE) from Mtb H37Rv strain induced TB patient-monocytes death after 4-h incubation, which was abrogated by neutralizing antibodies for TLR2 but not TLR4. Similarly, Pam3CSK4, a synthetic agonist triacylated ligand to TLR2 also induced death of monocytes, although it did not increase the cleaved caspase 9 level. Our findings suggest that monocytes from TB patients are more susceptible to die probably due to mitochondrial damage, and that cell death increases in the presence of mycobacterial antigen by a TLR2-dependent pathway.

MHC class I-restricted CD8 T lymphocyte epitopes are consisted of anchor motifs, TCR contact residues and the peptide backbone. A serial variant epitopes with substitution of amino acids at either anchor motifs or TCR contact residues have been synthesized for specific IFN-γ responses to clarify the TCR recognition mechanism as well as to assess the epitope prediction capacity of immunoinformatical programmes. CD8 T lymphocytes recognise the steric configuration of functional groups at the TCR contact side chain with a parallel observation that peptide backbones of various epitopes adapt to the conserved conformation upon binding to the same MHC class I molecule. Variant epitopes with amino acid substitutions at the TCR contact site are not recognised by specific CD8 T lymphocytes without compromising their binding capacity to MHC class I molecules, which demonstrates two discrete antigen presentation events for the binding of peptides to MHC class I molecules and for TCR recognition. The prediction outcome of immunoinformatical programmes is not consistent with results of epitope identification with laboratory experiments in the absence of information on the interaction with TCR contact residues. Immunoinformatical programmes based on the binding affinity to MHC class I molecules are not sufficient for the accurate prediction on CD8 T lymphocyte epitopes. The predictive capacity is further improved to distinguish mutant epitopes from the non-mutated one if the peptide-TCR interface is integrated into the computing simulation programme.

The observation that human monocytes cultured in the presence of the chemokine CCL18 showed increased survival (Wimmer A. et al., Blood. 2006, 108:3722-3729), led us to profile cytokine expression in CCL18-stimulated versus control cultures. CCL18 caused significantly increased expression of chemokines (CXCL8, CCL2, CCL3, and CCL22), IL-10 and PDGF, but no up-regulation of M1 cytokines IL-1β or IL-12. CCL18 stimulated monocytes matured into cells with morphological resemblance to IL-4 stimulated macrophages, and expressed the monocyte marker CD14 as well the M2 macrophage markers CD206 and 15-lipoxygenase, but no mature dendritic cell markers (CD80, CD83 or CD86). Functionally, CCL18-stimulated macrophages showed a high capacity for unspecific phagocytosis and for pinocytosis, which was not associated with an oxidative burst. These findings suggest that CCL18 activated macrophages stand at the cross-roads between inflammation and its resolution: The chemokines which are produced in response to CCL18 are angiogenic and attract various leukocyte populations, which sustain inflammation. However, the capacity of these cells to remove cellular debris without causing oxidative damage and the production of the anti-inflammatory IL-10 will initiate termination of the inflammatory response. In summary, CCL18 induces an M2 spectrum macrophage phenotype in the absence of IL-4.

Caspase-8 serves two paradoxical roles in T lymphocytes: it initiates apoptosis following death receptor engagement, and is also indispensible for proliferation following T cell antigen receptor (TCR) signaling. These opposing processes appear to be controlled by both spatial and quantitative differences in caspase-8 activation. Given differences in the turnover of T cell subsets, we compared caspase activity and susceptibility to cell death following TCR restimulation in murine CD4⁺ and CD8⁺αβ T cells and γδ T cells. We observed a spectrum of caspase activity in non-dying effector T cells in which CD4⁺ T cells manifested the lowest levels of active caspases whereas γδ T cells manifested the highest levels. Further analysis revealed that most of the difference in T cell subsets was due to high levels of active caspase-3 in non-dying effector γδ T cells. Despite this, γδ T cells manifested little spontaneous or CD3-restimulation induced cell death due to confinement of active caspases to the cell membrane. By contrast, CD4⁺ T cells were highly sensitive to CD3-induced cell death, associated with appearance of active caspases in the cytoplasm and cleavage of Bid and ICAD. Thus, the location and amount of active caspases distinguishes effector T cell subsets and profoundly influences the fate of the T cell response.

It is well established that T regulatory cells (Tregs) counteract tumor immunity. However, conflicting results describing the role of Tregs in hematological tumors warrant further investigations to clarify the interactions between Tregs and the tumor. B-cell malignancy derives from different stages of B-cell development and differentiation in which T-cells play a profound role. The transformed B-cell may still be in need of T-cell help to thrive but simultaneously they may be recognized and destroyed by cytotoxic lymphocytes. Recent reports demonstrate that Tregs can suppress and even kill B-cells as part of their normal function to rescue the body from autoimmunity. An emerging body of evidence points out that Tregs inhibit tumor-specific T-cells but may also have a role in suppressing the progression of the B-cell tumor. In this review, we discuss the origin and function of Tregs and their role in patients with B-cell tumors.

Recent scientific discoveries fuelled by the application of next-generation DNA and RNA sequencing technologies highlight the striking impact of these platforms in characterizing multiple aspects in genomics research. This technology has been used in the study of the B-cell and T-cell receptor repertoire. The novelty of immunosequencing comes from the recent rapid development of techniques and the exponential reduction in cost of sequencing. Here, we describe some of the technologies, which we collectively refer to as Rep-Seq (repertoire sequencing), to portray achievements in the field and to present the essential and inseparable role of next-generation sequencing to the understanding of entities in immune response. The large Rep-Seq data sets that should be available in the near future call for new computational algorithms to segue the transition from ‘classic’ molecular-based analysis to system-wide analysis. The combination of new algorithms with high-throughput data will form the basis for possible new clinical implications in personalized medicine and deeper understanding of immune behaviour and immune response.

Early studies that used parasite-infected interleukin-4 (IL-4) reporter animals led us to identify basophils as the primary source of IL-4 and hence propose the hypothesis that basophils trigger the development of antigen-specific T helper type 2 (Th2) immune responses in vivo. These findings appeared to resolve a long-standing puzzle underlying Th2 immunity, that is, ‘what is the source of the initial IL-4 necessary for CD4 T-cell differentiation into Th2 effector cells?’. However, results from extensive investigations of the contribution of basophils to Th2 immunity unveiled some controversial data that cast doubt on the initial hypothesis. In this review, the consensus and the controversy regarding the roles of basophils in infection and immunity, as well as outstanding questions for the future, are discussed.

Results from live-cell microscopy suggest that the behaviour of isolated components of the T-cell activation machinery in vitro does not represent the reality inside cells. Understanding the cellular-scale dynamics of microcluster migration can only be accomplished by in situ observation. Developments in ‘super-resolution’ microscopy have permitted investigators to move beyond tracking the movements of individual molecules, allowing the recognition of protein islands and nanodomains present in quiescent and active T cells. Many high-resolution techniques have their own susceptibilities to artefacts, so it is important to take a multifaceted approach to confirm results. A major challenge for the future will be to integrate all the new information into a coherent model of antigen recognition and T-cell activation.

B cells express two critical deaminases in the development of adaptive and innate immunity. Activation-induced cytidine deaminase (AID) functions in class switch recombination, somatic hypermutation and may result in affinity maturation of antibodies. Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G; A3G) is an innate anti-retroviral factor that inhibits HIV replication. We have studied a number of B-cell agonists with the aim of identifying the most effective agents that will up-regulate both deaminases and thereby enhance adaptive and innate immunity. CD40 ligand (CD40L) with interleukin-4 or HLA-class II antibodies significantly up-regulated both AID and A3G in isolated human CD19⁺ B cells. The functions of these deaminases were demonstrated by enhancement of B-cell surface expression of IgA and IgG and inducing significantly higher IgA and IgG4 antibodies. An enhanced A3G function was then demonstrated by inhibition of HIV-1 replication in co-culture of CD4⁺ T cells with autologous B cells, treated with CD40L and CD4 or HLA antibodies, compared with unstimulated human B cells. The dual B-cell-induced deaminase functions may be critical in IgA and IgG antibodies inhibiting pre-entry and A3G that of post-entry HIV-1 transmission and suggests a novel strategy of immunization, especially relevant to mucosal infections.

An efficacious tuberculosis (TB) vaccine will probably need to induce both CD4 and CD8 T-cell responses specific to a protective Mycobacterium tuberculosis antigen(s). To achieve this broad cellular immune response we tested a heterologous DNA/protein combination vaccine strategy. We used a purified recombinant protein preparation of a unique M. tuberculosis antigen (rMT1721) found in the urine of TB patients, an optimized plasmid DNA expressing this protein (DNA-MT1721), and a Toll-like receptor 4 agonist adjuvant. We found that priming mice with DNA-MT1721 and subsequently boosting with rMT1721 elicited high titres of specific IgG1 and IgG2a antibodies as well as high magnitude and polyfunctional CD4⁺ T-cell responses. However, no detectable CD8⁺ T-cell response was observed using this regimen of immunization. In contrast, both CD4⁺ and CD8⁺ T-cell responses were detected after a prime/boost vaccination regimen using rMT1721 as the priming antigen and DNA-MT1721 as the boosting immunogen. These findings support the exploration of heterologous DNA/protein immunization strategies in vaccine development against TB and possibly other infectious diseases.

CD300a, a membrane protein expressed on myeloid lineages and specific subsets of CD4⁺ T cells, has been reported to have inhibitory activities in cellular activation. However, the role of CD300a in Toll-like receptor (TLR) -mediated macrophage activation has not been investigated. The human monocytic cell lines THP-1 and U937 were stimulated with various TLR ligands after triggering of CD300a with specific monoclonal antibody. Interestingly, CD300a blocked TLR4-mediated and TLR9-mediated expression of pro-inflammatory mediators without affecting TLR3-mediated events. In contrast, CD300f, another member of the CD300 family, blocked the activation of cells induced by all TLR ligands. A transient transfection assay using luciferase reporter gene under the regulation of nuclear factor-κB binding sites indicated that co-transfection of CD300f blocked reporter expression induced by over-expression of both myeloid differentiation factor 88 (MyD88) and toll-interleukin 1 receptor-domain-containing adapter-inducing interferon-β (TRIF), whereas CD300a blocked only MyD88-induced events. Synthetic peptides representing immunoreceptor tyrosine-based inhibitory motifs of CD300a or CD300f mimicked the differential inhibition patterns of their original molecules. The use of various signalling inhibitors and Western blotting analysis revealed that TLR9/MyD88-mediated signalling was regulated mainly by SH2-containing tyrosine phosphatase 1 (SHP-1), which could be activated by CD300a or CD300f. In contrast, regulation of the TLR3/TRIF-mediated pathway required the combined action of SHP-1 and SHP-2, which could be accomplished by CD300f but not CD300a. These data indicate that CD300a and CD300f regulate the MyD88 and TRIF-mediated TLR signalling pathways through differential activation of SHP-1 and SHP-2.

Signal regulatory protein α (SIRPα/CD172a), expressed by myeloid cells including CD11b⁺ dendritic cells, interacts with ubiquitously expressed CD47 to mediate cell–cell signalling and therefore, may be pivotal in the development of tolerance or immunity. We show that in mice deficient in CD47 (CD47^−/−) the cellularity in gut-associated lymphoid tissues is reduced by 50%. In addition, the frequency of CD11b⁺ CD172a⁺ dendritic cells is significantly reduced in the gut and mesenteric lymph nodes, but not in Peyer’s patches. Activation of ovalbumin (OVA)-specific CD4⁺ T cells in the mesenteric lymph nodes after feeding OVA is reduced in CD47^−/− mice compared with wild-type however, induction of oral tolerance is maintained. The addition of cholera toxin generated normal serum anti-OVA IgG and IgA titres but resulted in reduced intestinal anti-OVA IgA in CD47^−/− mice. Replacing the haematopoietic compartment in CD47^−/− mice with wild-type cells restored neither the cellularity in gut-associated lymphoid tissues nor the capacity to produce intestinal anti-OVA IgA following immunization. This study demonstrates that CD47 signalling is dispensable for oral tolerance induction, whereas the expression of CD47 by non-haematopoietic cells is required for intestinal IgA B-cell responses. This suggests that differential CD4 T cell functions control tolerance and enterotoxin-induced IgA immunity in the gut.

We have previously reported that the 20-mer peptide p2340 (amino acids 2340–2359), of human thyroglobulin (Tg) has the unique feature that it causes experimental autoimmune thyroiditis (EAT) in mouse strains bearing high-responder (HR) or low-responder (LR) MHC haplotypes in Tg-induced EAT. In this study, we have employed fine epitope mapping to examine whether this property of p2340 is the result of recognition of distinct or shared minimal T-cell epitopes in the context of HR or LR MHC class II molecules. Use of overlapping peptides showed that a core minimal 9-mer epitope (LTWVQTHIR, amino acids 2344–2352) was recognized by p2340-primed T cells from both HR (H2^k,s) and LR (H2^b,d) strains, whereas a second 9-mer epitope (HIRGFGGDP, amino acids 2350–2358) was antigenic only in H2^s hosts. Truncation analysis of LTWVQTHIR and HIRGFGGDP peptides delineated them as the minimal epitopes recognized by p2340-primed T cells from the above strains. Subcutaneous challenge of all mouse strains with the 9-mer core peptide LTWVQTHIR in adjuvant elicited specific lymph node cell proliferative responses and mild EAT only in HR hosts, highlighting this sequence as a minimal pathogenic Tg peptide in EAT. The 9-mer peptide HIRGFGGDP was not found to be immunogenic in H2^s hosts. These data demonstrate that minimal T-cell epitopes, defined as autoantigenic in hosts of various MHC haplotypes, are not intrinsically immunogenic. Activation of naive autoreactive T cells may require contributions from flanking residues within longer peptide sequences encompassing these epitopes.