DEC205+ Dendritic Cell-Targeted Tolerogenic Vaccination Promotes Immune Tolerance in Experimental Autoimmune Arthritis.

Previous studies in mouse models of autoimmune diabetes and encephalomyelitis have indicated that the selective delivery of self-antigen to the endocytic receptor DEC205 on steady-state dendritic cells (DCs) may represent a suitable approach to induce Ag-specific immune tolerance. In this study, we aimed to examine whether DEC205(+) DC targeting of a single immunodominant peptide derived from human cartilage proteoglycan (PG) can promote immune tolerance in PG-induced arthritis (PGIA). Besides disease induction by immunization with whole PG protein with a high degree of antigenic complexity, PGIA substantially differs from previously studied autoimmune models not only in the target tissue of autoimmune destruction but also in the nature of pathogenic immune effector cells. Our results show that DEC205(+) DC targeting of the PG peptide 70-84 is sufficient to efficiently protect against PGIA development. Complementary mechanistic studies support a model in which DEC205(+) DC targeting leads to insufficient germinal center B cell support by PG-specific follicular helper T cells. Consequently, impaired germinal center formation results in lower Ab titers, severely compromising the development of PGIA. Overall, this study further corroborates the potential of prospective tolerogenic DEC205(+) DC vaccination to interfere with autoimmune diseases, such as rheumatoid arthritis.

Heat-shock proteins induce T-cell regulation of chronic inflammation.

Immune responses to certain heat-shock proteins (HSPs) develop in almost all inflammatory diseases; however, the significance of such responses is only now becoming clear. In experimental disease models, HSPs can prevent or arrest inflammatory damage, and in initial clinical trials in patients with chronic inflammatory disease, HSP-derived peptides have been shown to promote the production of anti-inflammatory cytokines, indicating that HSPs have immunoregulatory potential. In this Review, we discuss the unique characteristics of HSPs that endow them with these immunoregulatory qualities.

Regulatory T cells that recognize a ubiquitous stress-inducible self-antigen are long-lived suppressors of autoimmune arthritis.

Reestablishing self-tolerance in autoimmunity is thought to depend on self-reactive regulatory T cells (Tregs). Exploiting these antigen-specific regulators is hampered by the obscure nature of disease-relevant autoantigens. We have uncovered potent disease-suppressive Tregs recognizing Heat Shock Protein (Hsp) 70 self-antigens, enabling selective activity in inflamed tissues. Hsp70 is a major contributor to the MHC class II ligandome. Here we show that a conserved Hsp70 epitope (B29) is present in murine MHC class II and that upon transfer, B29-induced CD4(+)CD25(+)Foxp3(+) T cells suppress established proteoglycan-induced arthritis in mice. These self-antigen-specific Tregs were activated in vivo, and when using Lymphocyte Activation Gene-3 as a selection marker, as few as 4,000 cells sufficed. Furthermore, depletion of transferred Tregs abrogated disease suppression. Transferred cells exhibited a stable phenotype and were found in joints and draining lymph nodes up to 2 mo after transfer. Given that (i) B29 administration by itself suppressed disease, (ii) our findings were made with wild-type (T-cell receptor nontransgenic) Tregs, and (iii) the B29 human homolog is presented by HLA class II, we are nearing translation of antigen-specific Treg activation as a promising intervention for chronic inflammatory diseases.

Heat shock proteins can be targets of regulatory T cells for therapeutic intervention in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by excessive immune responses resulting in inflammation of the joints. Although current therapies can be successful in dampening inflammation, a long-lived state of tolerance is seldom achieved. Therefore, novel therapies are needed that restore and maintain tolerance in patients with RA. Targeting regulatory T cells (Tregs) is a successful strategy to achieve tolerance, as was shown in studies performed in animal models and in human clinical trials. The antigen-specificity of Tregs is crucial for their effectiveness and allows for very specific targeting of these cells. However, which antigen is suitable for autoimmune diseases such as RA, for which the autoantigens are largely unknown? Heat shock proteins (HSPs) are ubiquitously expressed and can be up-regulated during inflammation. Additionally, HSPs, or HSP-derived peptides are immunogenic and can be recognised by a variety of immune cells, including Tregs. Therefore, this review highlights the potential of HSP-specific Tregs to control inflammatory immune responses. Targeting HSP-specific Tregs in RA can be achieved via the administration of HSPs (derived peptides), thereby controlling inflammatory responses. This makes HSPs attractive candidates for therapeutic intervention in chronic autoimmune diseases, with the ultimate goal of inducing long-lasting tolerance.

Autoreactive HSP60 epitope-specific T-cells in early human atherosclerotic lesions.

Atherosclerosis is a multifactorial chronic inflammatory disease characterized by the presence of T-cells, macrophages, and dendritic cells in the arterial intima. Classical risk factors lead to over-expression of stress proteins, especially heat shock protein 60 (HSP60). HSP60 on the surface of arterial endothelial cells (ECs) then becomes a target for pre-existing adaptive anti-HSP60 immunity resulting in infiltration of the intima by mononuclear cells. In the present study, T-cells derived from early, clinically still inapparent human atherosclerotic lesions were analyzed phenotypically and for their reactivity against HSP60 and HSP60-derived peptides. HSP60 was detected in ECs and CD40- and HLA Class II-positive cells within the intima. Effector memory CD4(+) T-cells producing high amounts of interferon-γ and low levels of interleukin-4 were the dominant subpopulation. T-cells derived from late lesions displayed a more restricted T-cell receptor repertoire to HSP60-derived peptides than those isolated from early lesions. Increased levels of soluble HSP60 and circulating anti-human HSP60 autoantibodies were found in donors with late but not early lesions. This is the first functional study of T-cells derived from early human atherosclerotic lesions that supports the previously proposed concept that HSP60-reactive T-cells initiate atherosclerosis by recognition of atherogenic HSP60 epitopes.

CD30 discriminates heat shock protein 60-induced FOXP3+ CD4+ T cells with a regulatory phenotype.

In many animal models, the manifestations of inflammatory diseases can be prevented by the adoptive transfer of CD4(+)FOXP3(+) regulatory T cells (Tregs). CD4(+)FOXP3(+) Tregs can be obtained by isolation and expansion of polyclonal naturally occurring Tregs or by Ag-specific activation of CD4(+)CD25(-)FOXP3(-) T cells. Two major obstacles are hampering the translation of this latter protocol into therapeutic application. First, there is a lack of knowledge on relevant autoantigens. Second, the resulting population is contaminated with activated CD4(+) T cells that transiently express Forkhead box P3 but gain no regulatory function. Therefore, these cells may not be safe for clinical application. In this study, we demonstrate that highly suppressive FOXP3(+) Tregs can be induced in vitro by the activation of CD4(+)CD25(-) T cells with the self-Ag human 60-kDa heat shock protein (HSP60). The activation induced suppressive FOXP3(+) Tregs can be distinguished by surface expression of CD30 from nonsuppressive FOXP3(+) effector cells. We confirm that the induced CD30(+)FOXP3(+) Tregs recognize HSP60 epitopes and that the induction of Tregs by HSP60 is enhanced by signaling via TLR4 on APCs. These findings have implications for the generation and isolation of pure populations of Ag-specific Tregs, with the potential to prevent and treat human inflammatory diseases.

Dynamics of heat shock protein 60 in endothelial cells exposed to cigarette smoke extract.

Heat shock protein 60 (HSP60), expressed on the surface of endothelial cells (ECs) stressed by e.g. oxidized LDL or mechanical shear, was shown to function as an auto-antigen and thus as a pro-atherosclerotic molecule. The aim of this study was to determine whether cigarette smoke chemicals can lead to the activation of the "HSP60 pathway." It was also our aim to elucidate the dynamics of HSP60 from gene expression to endothelial surface expression and secretion. Here we show for the first time that the exposure of human umbilical vein endothelial cells (HUVECs) to cigarette smoke extract (CSE) results in an up-regulation of HSP60 mRNA. Live cell imaging analysis of a HSP60-EYFP fusion protein construct transfected into ECs revealed that mitochondrial structures collapse in response to CSE exposure. As a result, HSP60 is released from the mitochondria, transported to the cell surface, and released into the cell culture supernatant. Analysis of HSP60 in the sera of healthy young individuals exposed to secondhand smoke revealed significantly elevated levels of HSP60. Cigarette smoking is one of the most relevant risk factors for atherosclerosis. Herein, we provide evidence that cigarette smoke may initiate atherosclerosis in the sense of the "auto-immune hypothesis of atherosclerosis."

A novel heat-shock protein coinducer boosts stress protein Hsp70 to activate T cell regulation of inflammation in autoimmune arthritis.

OBJECTIVE: Stress proteins, such as members of the heat-shock protein (HSP) family, are up-regulated by cells in inflamed tissue and can be viewed functionally as "biomarkers" for the immune system to monitor inflammation. Exogenous administration of stress proteins has induced immunoregulation in various models of inflammation and has also been shown to be effective in clinical trials in humans. This study was undertaken to test the hypothesis that boosting of endogenous HSP expression can restore effective immunoregulation through T cells specific for stress proteins. METHODS: Stress protein expression was manipulated in vivo and in vitro with a food component (carvacrol), and immune recognition of stress proteins was studied. RESULTS: Carvacrol, a major compound in the oil of many Origanum species, had a notable capacity to coinduce cellular Hsp70 expression in vitro and, upon intragastric administration, in Peyer's patches of mice in vivo. As a consequence, carvacrol specifically promoted T cell recognition of endogenous Hsp70, as demonstrated in vitro by the activation of an Hsp70-specific T cell hybridoma and in vivo by amplified T cell responses to Hsp70. Carvacrol administration also increased the number of CD4+CD25+FoxP3+ T cells, systemically in the spleen and locally in the joint, and almost completely suppressed proteoglycan-induced experimental arthritis. Furthermore, protection against arthritis could be transferred with T cells isolated from carvacrol-fed mice. CONCLUSION: These findings illustrate that a food component can boost protective T cell responses to a self stress protein and down-regulate inflammatory disease, i.e., that the immune system can respond to diet.

Interleukin-4 therapy of psoriasis induces Th2 responses and improves human autoimmune disease.

Selective skewing of autoreactive interferon-gamma (IFN-gamma)-producing T helper cells (Th1) toward an interleukin-4 (IL-4)-producing (Th2) phenotype can in experimental animals alleviate autoimmune disease without inducing general immunosuppression. In a prospective dose escalation study, we assessed treatment with human IL-4 (rhuIL-4) in 20 patients with severe psoriasis. The therapy was well tolerated, and within six weeks all patients showed decreased clinical scores and 15 improved more than 68%. Stable reduction of clinical scores was significantly better at 0.2-0.5 microg rhuIL-4 than at < or =0.1 microg rhuIL-4 (P = 0.009). In psoriatic lesions, treatment with 0.2-0.5 microg/kg rhuIL-4 reduced the concentrations of IL-8 and IL-19, two cytokines directly involved in psoriasis; the number of chemokine receptor CCR5+ Th1 cells; and the IFN-gamma/IL-4 ratio. In the circulation, 0.2-0.5 microg/kg rhuIL-4 increased the number of IL-4+CD4+ T cells two- to three-fold. Thus, IL-4 therapy can induce Th2 differentiation in human CD4+ T cells and has promise as a potential treatment for psoriasis, a prototypic Th1-associated autoimmune disease.

Molecular mimicry between Helicobacter pylori antigens and H+, K+ --adenosine triphosphatase in human gastric autoimmunity.

Autoimmune gastritis and Helicobacter pylori-associated gastric atrophy develop through similar mechanisms involving the proton pump H+,K+-adenosine triphosphatase as autoantigen. Here, we report that H. pylori-infected patients with gastric autoimmunity harbor in vivo-activated gastric CD4+ T cells that recognize both H+, K+-adenosine triphosphatase and H. pylori antigens. We characterized the submolecular specificity of such gastric T cells and identified cross-reactive epitopes from nine H. pylori proteins. Cross-reactive H. pylori peptides induced T cell proliferation and expression of T helper type 1 functions. We suggest that in genetically susceptible individuals, H. pylori infection can activate cross-reactive gastric T cells leading to gastric autoimmunity via molecular mimicry.

Intradermal injection of Hsp60 induces cytokine responses in canine atopic and healthy skin.

The purpose of this study was to investigate the immunoregulatory potential of Hsp60 in the skin of dogs with atopic dermatitis. Three dogs with chronic atopic dermatitis and four healthy dogs were injected intradermally with Hsp60 and phosphate-buffered saline. Biopsies were taken before testing from non-injected control skin, lesional and non-lesional atopic skin, and 48 and 72 h after injection. Analysis of cytokine messenger RNA was performed using quantitative real-time polymerase chain reaction. Forty-eight hours after Hsp60 injection, a rise in interleukin (IL)-10 was found (P = 0.034) with the highest expression levels in non-lesional atopic and control skin. A rise of transforming growth factor beta (P = 0.015) and IL-12p40 (P = 0.017) was noticed 72 h after Hsp60 injection in control skin. No significant differences were observed for the expression of IL-4, IL-12p35, and interferon gamma. The results indicate that Hsp60 is able to induce cytokines of a regulatory and Th1 phenotype in the skin. Furthermore, this study seems to provide a first indication of deficient Hsp60 response in atopic dermatitis affected skin.

In vivo imaging of the effect of LPS on arterial endothelial cells: molecular imaging of heat shock protein 60 expression.

Bacterial endotoxins are known as stress factors for endothelial cells. In 20 normocholesterolemic New Zealand White (NZW) rabbits, endothelial stress was induced by intravenous (i.v.) injection of lipopolysaccharide (LPS), while eight NZW rabbits were sham-treated or served as untreated controls. In vivo molecular imaging was performed using co-registered computer tomography and positron emission tomography 24 h after i.v. injection of (124)I-labeled monoclonal anti-HSP60 or (124)I-radiolabelled isotype control antibodies. Compared to control animals, in vivo images of rabbit aortae revealed significantly increased endothelial binding of (124)I-labeled anti-HSP60 antibodies upon LPS, especially at sites of aortal branching. This was confirmed by immunohistochemistry and autoradiography data. Our results showed, as proof-of-principle, that HSP60-expression in normocholesterolemic rabbits is significantly increased after induction of endothelial stress and that non-invasive in vivo molecular imaging of early aortal HSP60-expression using (124)I-labeled anti-HSP60 monoclonal antibodies is possible.

Lactobacillus rhamnosus GG-Derived Soluble Mediators Modulate Adaptive Immune Cells.

Probiotics and probiotic-related nutritional interventions have been described to have beneficial effects on immune homeostasis and gut health. In previous studies, Lactobacillus rhamnosus GG (LGG) soluble mediators (LSM) have been demonstrated to exert beneficial effects in preclinical models of allergic sensitization, bacterial infection, and intestinal barrier function. In the context of allergic diseases, differentiation of dendritic cells (DCs) and their interactions with T cell populations are crucial for driving tolerogenic responses. In this study, we set out to evaluate whether these LSM can modulate DC maturation and have an impact on prompting protective and/or tolerogenic T cell responses. Monocytes were isolated from PBMC of healthy blood donors and cultured in the presence of GM-CSF, IL-4, and LSM or unconditioned bacterial culture medium control (UCM) during 6 days to induce DC differentiation. Subsequently, these DCs were matured in the presence of TNF-α for 1 day and analyzed for their phenotype and ability to induce autologous T cell activation and differentiation to model recall antigens. After 7 days of co-culture, T cells were analyzed for activation and differentiation by flow cytometry of intracellular cytokines (IFN-γ, IL-2, IL-10, and IL-17A), activation markers (CD25), and Foxp3+ expression. LSM did not alter DC numbers or maturation status. However, these DCs did show improved capacity to induce a T cell response as shown by increased IL-2 and IFN-γ producing T cell populations upon stimulation with recall antigens. These enhanced recall responses coincided with enhanced Foxp3+ expression that was not observed when T cells were cultured in the presence of UCM-treated DCs. By contrast, the number of activated T cells (determined by CD25 expression) was only slightly increased. In conclusion, this study reveals that LSM can influence adaptive immune responses as shown by the modulation of DC functionality. These mechanisms might contribute to previous observed effects in animal models in vivo. Altogether, these results suggest that LSM may provide an alternative to live probiotics in case life bacteria may not be used because of health conditions, although further clinical testing is needed.

Routing dependent immune responses after experimental R848-adjuvated vaccination.

Most traditional vaccines are administered via the intramuscular route. Other routes of administration however, can induce equal or improved protective memory responses and might provide practical advantages such as needle-free immunization, dose sparing and induction of tissue-specific (mucosal) immunity. Here we explored the differences in immunological outcome after immunization with model antigens via two promising immunization routes (intradermal and intranasal) with or without the experimental adjuvant and TLR7/8-agonist R848. Because the adaptive immune response is largely determined by the local innate cells at the site of immunization, the effect of R848-adjuvation on local cellular recruitment, antigenic uptake by antigen-presenting cells and the initiation of the adaptive response were analyzed for the two routes of administration. We show a general immune-stimulating effect of R848 irrespective of the route of administration. This includes influx of neutrophils, macrophages and dendritic cells to the respective draining lymph nodes and an increase in antigen-positive antigen-presenting cells which leads for both intradermal and intranasal immunization to a mainly TH1 response. Furthermore, both intranasal and intradermal R848-adjuvated immunization induces a local shift in DC subsets; frequencies of CD11b+DC increase whereas CD103+DC decrease in relative abundance in the draining lymph node. In spite of these similarities, the outcome of immune responses differs for the respective immunization routes in both magnitude and cytokine profile. Via the intradermal route, the induced T-cell response is higher compared to that after intranasal immunization, which corresponds with the local higher uptake of antigen by antigen-presenting cells after intradermal immunization. Furthermore, R848-adjuvation enhances ex vivo IL-10 and IL-17 production after intranasal, but not intradermal, T-cell activation. Quite the opposite, intradermal immunization leads to a decrease in IL-10 production by the vaccine induced T-cells. This knowledge may lead to a more rational development of novel adjuvanted vaccines administered via non-traditional routes.

Cell stress induced HSP are targets of regulatory T cells: a role for HSP inducing compounds as anti-inflammatory immuno-modulators?

T cell responses to heat shock proteins (HSP) have disease suppressive activities through production of anti-inflammatory cytokines in patients and in models of inflammatory diseases. There is evidence that the anti-inflammatory activity of HSP-specific T cells depends on their recognition of endogenous HSP epitopes as expressed by stressed cells at sites of inflammation. Previously, we have demonstrated that such T cells can be induced by conserved sequences of microbial HSP. Now we propose that drug induced up-regulation of endogenous HSP can contribute to anti-inflammatory T cell regulation.

The Enigma of Heat Shock Proteins in Immune Tolerance.

The fundamental problem of autoimmune diseases is the failure of the immune system to downregulate its own potentially dangerous cells, which leads to destruction of tissue expressing the relevant autoantigens. Current immunosuppressive therapies offer relief but fail to restore the basic condition of self-tolerance. They do not induce long-term physiological regulation resulting in medication-free disease remissions. Heat shock proteins (HSPs) have shown to possess the capacity of inducing lasting protective immune responses in models of experimental autoimmune diseases. Especially mycobacterial HSP60 and HSP70 were shown to induce disease inhibitory IL-10-producing regulatory T cells in many different models. This in itself may seem enigmatic, since based on earlier studies, HSPs were also coined sometimes as pro-inflammatory damage-associated molecular patterns. First clinical trials with HSPs in rheumatoid arthritis and type I diabetes have also indicated their potential to restore tolerance in autoimmune diseases. Data obtained from the models have suggested three aspects of HSP as being critical for this tolerance promoting potential: 1. evolutionary conservation, 2. most frequent cytosolic/nuclear MHC class II natural ligand source, and 3. upregulation under (inflammatory) stress. The combination of these three aspects, which are each relatively unique for HSP, may provide an explanation for the enigmatic immune tolerance promoting potential of HSP.

Bystander activation of irrelevant CD4+ T cells following antigen-specific vaccination occurs in the presence and absence of adjuvant.

Autoimmune and other chronic inflammatory diseases (AID) are prevalent diseases which can severely impact the quality of life of those that suffer from the disease. In most cases, the etiology of these conditions have remained unclear. Immune responses that take place e.g. during natural infection or after vaccination are often linked with the development or exacerbation of AID. It is highly debated if vaccines induce or aggravate AID and in particular adjuvants are mentioned as potential cause. Since vaccines are given on a large scale to healthy individuals but also to elderly and immunocompromised individuals, more research is warranted. Non-specific induction of naïve or memory autoreactive T cells via bystander activation is one of the proposed mechanisms of how vaccination might be involved in AID. During bystander activation, T cells unrelated to the antigen presented can be activated without (strong) T cell receptor (TCR) ligation, but via signals derived from the ongoing response directed against the vaccine-antigen or adjuvant at hand. In this study we have set up a TCR transgenic T cell transfer mouse model by which we were able to measure local bystander activation of transferred and labeled CD4+ T cells. Intramuscular injection with the highly immunogenic Complete Freund's Adjuvant (CFA) led to local in vivo proliferation and activation of intravenously transferred CD4+ T cells in the iliac lymph node. This local bystander activation was also observed after CFA prime and Incomplete Freund's Adjuvant (IFA) boost injection. Furthermore, we showed that an antigen specific response is sufficient for the induction of a bystander activation response and the general, immune stimulating effect of CFA or IFA does not appear to increase this effect. In other words, no evidence was obtained that adjuvation of antigen specific responses is essential for bystander activation.

Dynamics of APC recruitment at the site of injection following injection of vaccine adjuvants.

Vaccines often contain adjuvants to strengthen the response to the vaccine antigen. However, their modes of action at the site of injection (SOI) are poorly understood. Therefore, we assessed the local effects of adjuvant on the innate immune system in mice. We investigated the safe, widely used adjuvants MF59 and aluminum hydroxide (alum), as well as trehalose-6,6'-dibehenate (TDB), Complete Freund's Adjuvant (CFA) and the Toll-Like-Receptor-ligands lipopolysaccharide (LPS) and Pam3CysSerLys4 (Pam3CSK4). We assessed muscle immune cell infiltration after adjuvant injection and observed 16h post immunization (hpi) an increased influx with CFA, MF59 and TDB, but not with alum, LPS or Pam3CSK4. An elevated influx with the latter three became visible only 72hpi. Contribution of granulocytes, macrophages and dendritic cells to the influx differed per adjuvant and in time. Adjuvants generally induced a local pro-inflammatory micro-milieu that was transient except for CFA and TDB. The gene expression of CXCL-1, CCL-2 and CCL-5, involved in recruitment of immune cells, varied per adjuvant and corresponded grossly with the observed influx of granulocytes and monocytes/macrophages. Muscles injected with CFA or MF59 (when co-injected with peptide) resulted in APC ex vivo capable to induce proliferation of peptide-specific T-cells. By adding in vitro an excess of peptide to the APC/T cell co-cultures, we observed an adjuvant-enhanced co-stimulation or antigen presentation by APC after CFA- but not MF59-injection. After TDB-injection this effect was observed only at 72hpi, but not 24hpi. Thus the cellular influx profile and the local cytokine and chemokine micro-milieu in the muscle were strongly influenced by the type of adjuvant. Additionally, the capacity of muscle APC to load and present antigen was affected by the adjuvant. These findings may assist the development of novel adjuvanted vaccines in a more rational manner.

Regulatory T cell frequencies and phenotypes following anti-viral vaccination.

Regulatory T cells (Treg) function in the prevention of excessive inflammation and maintenance of immunological homeostasis. However, these cells may also interfere with resolution of infections or with immune reactions following vaccination. Effects of Treg on vaccine responses are nowadays investigated, but the impact of vaccination on Treg homeostasis is still largely unknown. This may be a relevant safety aspect, since loss of tolerance through reduced Treg may trigger autoimmunity. In exploratory clinical trials, healthy adults were vaccinated with an influenza subunit vaccine plus or minus the adjuvant MF59®, an adjuvanted hepatitis B subunit vaccine or a live attenuated yellow fever vaccine. Frequencies and phenotypes of resting (rTreg) and activated (aTreg) subpopulations of circulating CD4+ Treg were determined and compared to placebo immunization. Vaccination with influenza vaccines did not result in significant changes in Treg frequencies and phenotypes. Vaccination with the hepatitis B vaccine led to slightly increased frequencies of both rTreg and aTreg subpopulations and a decrease in expression of functionality marker CD39 on aTreg. The live attenuated vaccine resulted in a decrease in rTreg frequency, and an increase in expression of activation marker CD25 on both subpopulations, possibly indicating a conversion from resting to migratory aTreg due to vaccine virus replication. To study the more local effects of vaccination on Treg in lymphoid organs, we immunized mice and analyzed the CD4+ Treg frequency and phenotype in draining lymph nodes and spleen. Vaccination resulted in a transient local decrease in Treg frequency in lymph nodes, followed by a systemic Treg increase in the spleen. Taken together, we showed that vaccination with vaccines with an already established safe profile have only minimal impact on frequencies and characteristics of Treg over time. These findings may serve as a bench-mark of inter-individual variation of Treg frequencies and phenotypes following vaccination.

Heat shock protein 60: identification of specific epitopes for binding to primary macrophages.

In the present study, we characterized regions of human heat shock protein (HSP) 60 responsible for binding to primary macrophages. Studies using 20-mer peptides of the HSP60 sequence to compete with HSP60-binding to macrophages from C57BL/6J mice showed that regions aa241-260, aa391-410 and aa461-480 are involved in surface-binding. HSP60 mutants, lacking the N-terminal 137, 243 or 359 amino acids, inhibited HSP60-binding to primary macrophages to different degrees, demonstrating that all three regions are required for optimal binding. Analysis of different pro- and eukaryotic HSP60 species indicated that phylogenetically separate HSP60 species use different binding sites on primary macrophages.