2A and B). Thus, each dose of α-GalCer

adjuvant delivered by the intranasal route resulted in the activation and expansion of NKT cells with IFN-γ producing potential along with an increase in activated DCs. On the other hand, a second dose of α-GalCer administered by the intravenous route resulted in only a slight increase in NKT cell proliferation, with no concurrent increase in IFN-γ production by NKT cells and no increase in activated DCs. Finally, the significant increase in the activation and reactivation of NKT cells and DCs from the booster immunization by the intranasal route with α-GalCer+OVA also translated into significant increases in antigen-specific cytotoxic T lymphocyte (CTL) activity and IFN-γ-producing cells after the booster dose, which was not observed after the intravenous booster immunization (Fig. 2C and D respectively). Since the primary immunization with α-GalCer+OVA resulted in the expansion buy TSA HDAC of NKT cells that peaked at day 5 in the lung and did not decrease to base-line levels even at day 10 post-immunization (Fig. 1D),

we evaluated whether the second increase in NKT cells is a consequence of the continued effect of the priming dose of α-GalCer or the effectiveness of the second dose delivered on day 5. For this, we delayed the booster immunization until day 23 post-priming and characterized NKT cells and DCs in different tissues on days 24, 26, and 28 (i.e. days 1, 3, and 5 respectively, click here relative to the booster dose, Fig. 3A). Significant increases in the percentages of IFN-γ-producing NKT cells were observed in the spleen and lung of mice immunized with the booster dose of α-GalCer+OVA at day 24 (i.e. day 1 after the booster immunization, Fig. 3B) and furthermore, significant expansion of NKT cells was observed in the lung between days 1 and 5 after the booster immunization (Fig.

3D) compared with that in either the OVA only control group of mice or those that received only the priming dose of α-GalCer+OVA. We also found CD11c+ DCs expressing SSR128129E slightly increased levels of the CD86 activation marker on day 24 (i.e. day 1 after the booster dose), when compared with the DCs from mice in the OVA control group (Fig. 3F). These results from mice that received the priming and boosting doses of α-GalCer+OVA by the intranasal route 23 days apart (the longer immunization scheme) were similar to those observed when the two doses were delivered 5 days apart (the shorter immunization scheme). Thus, regardless of the timing of the second dose, α-GalCer administration by the intranasal route leads to repeated activation of NKT cells, primarily in the lung. These results employing α-GalCer as an adjuvant delivered by the intranasal route are in contrast to those where primary and booster immunizations of α-GalCer+OVA delivered by the intravenous route 23 days apart.

These findings altogether suggested that TGF-β-expressing immature AE-pe-DCs might play a significant role in the generation of a regulatory immune response within the peritoneal cavity of AE-infected mice. Alveolar echinococcosis (AE) is a severe chronic helminthic disease accidentally affecting humans. Following infection by peroral uptake of Echinococcus multilocularis eggs, AE develops as a consequence of intrahepatic establishment of the larval stage (= metacestode) of the tapeworm. From the liver, the metacestode spreads to other organs by

infiltration or metastasis formation, thus clinically AE rather resembles a tumour-like disease. The natural intermediate hosts involved in the life cycle of the parasite are predominantly small rodents. Therefore, the laboratory mouse is an excellent model to study the host–parasite interplay. Tyrosine Kinase Inhibitor Library mouse Experimentally, intraperitoneal inoculation of metacestode vesicles is referred to as secondary infection. In the peritoneal cavity of metacestode-infected mice [AE-mice], inter-visceral tumour-like growth of the metacestode overcomes the immune system such as to establish a chronic

phase of infection, which persists approximately between 2 and 6 months p.i. By the end of this time period, infection/disease reaches a terminal stage where mice have to be sacrificed because of severity of symptoms. In the host–parasite interplay, metacestode surface molecules as well as excretory/secretory (E/S) products are considered as important key players (1). The intraperitoneal murine model https://www.selleckchem.com/products/dinaciclib-sch727965.html of AE offers the opportunity to study the direct effect of metacestodes on periparasitic peritoneal cells, including especially dendritic cells (DCs), the most important antigen-presenting cells (APC) in the initiation of a Th1- or Th2-oriented immune response. Several studies so far suggested that distinct subsets

of DCs differentially modulate T-helper responses, but other studies pointed to a dominant role for microbial stimuli and the local microenvironment in this process (2). In the frame of a Th1 immune orientation, it is largely accepted that DCs are activated mostly by bacterial or viral pathogens via toll-like receptor (TLR) ligation to produce IL-12 and TNF-α, both pro-inflammatory cytokines inducing a Th1-oriented response (3,4). Th1-associated DC activation by microbial products evokes 4-Aminobutyrate aminotransferase rapid phenotypic changes, including up-regulation of surface markers for DC maturation such as MHC class II, CD80, CD86 and CD40 molecules (5,6). How DCs elicit a Th2 response is more controversial. There is no mirror image signature of cytokine and surface ligands that DCs express to stimulate Th2 differentiation. Some examples of helminth antigens, including the products of filarial Acanthocheilonema viteae (ES-62) (7), Schistosoma mansoni soluble egg antigen (SEA) (8) and the schistosome-associated glycan lacto-N-ficopentaose III (LNFPIII) (9), do not appear to induce IL-12 production by DCs (8,10).

Consequently, a mechanism by which p21Cip1 binds to and inhibits AP-1 components should not block the ability of anergic Th1 cells to proliferate in response to exogenous IL-2 in secondary n-butyrate-free cultures. In contrast to anergic Th1 cells, Deforolimus there was no p21Cip1 in control Th1 cells before restimulation.

p21Cip1 gradually accumulated in the control Th1 cells, demonstrating very low levels at the early time periods at which p-JNK or p-c-jun were up-regulated in response to antigen restimulation. Therefore, in the control Th1 cells, early activation events were completed before p21Cip1 reached detectable levels, possibly explaining why p21Cip1 did not block initial cell division in control Th1 cells unlike the anergic Th1 cells. In the immunoprecipitation experiments, most of the JNK in the cell lysates did not associate with p21Cip1 except for a small amount in the anergic Th1 cells restimulated for 2 hr. Normally, only a small portion of JNK present in the cell becomes phosphorylated upon T-cell receptor stimulation. As the JNK antibody used in this study recognizes p-JNK as well as unphosphorylated JNK, the thin band of JNK that was associated with p21Cip1 in the restimulated anergic group could represent the phosphorylated form of JNK. p21Cip1 interaction with p-JNK and p-c-jun was demonstrated

in this study. It is not clear why p21Cip1 would bind preferentially to the phosphorylated forms of these FK228 order proteins, but phosphorylation-dependent confirmation changes may be in effect regulating this interaction. This interaction was confirmed in reciprocal immunoprecipitations. Unlike p21Cip1, p27Kip1 did not seem to associate with the MAPK in the anergic Th1 cells. p27Kip1 has been suggested to be a mediator of Adenosine T-cell tolerance in a study of human alloantigen-specific T-cell tolerance in which over-expression of p27Kip1 in primary cultures was shown to result in unresponsiveness in T-cell clones upon rechallenge in secondary cultures.3 In addition, p27Kip1 was recently shown to be required for transplantation tolerance induced

in vivo by costimulation blockade.38 Yet in one study, the role of p27Kip1 in T-cell anergy was questioned by investigators who showed that anti-TCR antibody could induce tolerance in p27Kip1-deficient CD4+ T cells in vitro.39 In our model, anergy induced by exposure to HDAC inhibitors, known to be potent stimulators of p21Cip1, seems to primarily rely on this CDK inhibitor rather than p27Kip1. The levels of p27Kip1 were not higher in the anergic Th1 cells than control Th1 cells at the end of 6-day primary cultures. p27Kip1 down-regulated rather than up-regulated in T cells treated with antigen and n-butyrate appeared to contradict reports in the literature describing an increase in p27Kip1 following exposure to n-butyrate.

The placental vascular dysfunction does extend to other fetal vascular beds including endothelial cells from umbilical vessels, where there are reports of elevated basal iNOS activity and altered sensitivity to insulin. There is emerging evidence of epigenetic modulation of fetal endothelial selleck screening library genes in diabetes and long-term vascular consequences

of this. Thus, placental vascular dysfunction in diabetes may be contributing to and describing disturbances in the fetal vasculature, which may produce an overt pathological response in later life if challenged with additional cardiovascular stresses. “
“Please cite this paper as: Arkill KP, Neal CR, Mantell JM, Michel CC, Qvortrup K, Rostgaard J, Bates DO, Knupp C, Squire JM. 3D reconstruction of the glycocalyx structure in mammalian capillaries using electron tomography. Microcirculation 19: 343–351, 2012. Objective:  Visualising the molecular strands making up the glycocalyx in the lumen of small blood vessels has proved to be difficult using conventional transmission electron microscopy techniques. Images obtained from tissue stained in a variety of ways have revealed a regularity in the organisation of the proteoglycan components of the glycocalyx layer (fundamental spacing about 20 nm), but require a large sample number. Attempts to visualise the glycocalyx face-on (i.e. in a direction perpendicular to the endothelial cell layer in the Wnt inhibitors clinical trials lumen and directly

applicable for permeability modelling) has had limited success (e.g. freeze fracture). A new approach is therefore needed. Methods:  Here we demonstrate the effectiveness of using the relatively novel electron microscopy technique of 3D electron tomography on two differently stained glycocalyx preparations. A tannic acid staining

method and a novel staining technique using Lanthanum Dysprosium Glycosamino Glycan adhesion (the LaDy GAGa method). Results:  3D electron tomography reveals details of the architecture of the glycocalyx just above the endothelial cell layer. The LaDy GAGa method visually appears to show more complete coverage and more depth than the Tannic Acid staining method. Conclusion:  The tomographic reconstructions show a potentially significant improvement in determining aminophylline glycocalyx structure over standard transmission electron microscopy. “
“Please cite this paper as: Ella, Yang, Clifford, Gulia, Dora, Meininger, Davis and Hill (2010). Development of an Image-Based System for Measurement of Membrane Potential, Intracellular Ca2+ and Contraction in Arteriolar Smooth Muscle Cells. Microcirculation17(8), 629–640. Objective:  Changes in smooth muscle cell (SMC) membrane potential (Em) are critical to vasomotor responses. As a fluorescent indicator approach would lessen limitations of glass electrodes in contracting preparations, we aimed to develop a Forster (or fluorescence) resonance energy transfer (FRET)-based measurement for Em.

78 Similarly, other purified TLR agonists and inflammatory cytokines that induce the maturation of dendritic cells and augment expression of cell surface molecules that promote T-cell stimulation (e.g. CD80, CD86 and MHC) have also been reported to override Treg-cell suppression through IL-6-independent pathways.79–81 Even in the absence of APCs, cell-intrinsic stimulation through defined TLRs can also trigger shifts in Treg-cell suppression. For example, purified TLR2 agonists stimulate reductions in suppressive potency for mouse Treg cells, and TLR8 agonists trigger similar reductions in potency for human Treg cells.82–84

On the other hand, microbial ligands can also augment Treg-suppressive potency. Mouse CD25+ Treg cells selectively express TLR4,

and lipopolysaccharide stimulation augments their suppressive potency;85 whereas flagellin stimulation via C646 ic50 TLR5 augments the suppressive potency of human Treg cells.86 Taken together, these in vitro studies illustrate the enormous potential whereby microbes and the response to infection can influence immune activation through shifts in Treg-cell suppression. The cumulative impacts whereby pathogens that express multiple TLR ligands and the ensuing immune response on shifts in Treg-suppressive potency have also been characterized for green fluorescent protein-positive (GFP+) cells recovered from Foxp3GFP reporter mice directly ex vivo following infection.87 For example, at Smoothened antagonist relatively early time-points during persistent Salmonella infection, when the activation of effector T cells is blunted and the pathogen burden is progressively increasing, the suppressive potency for GFP+ Treg cells is augmented.59 Conversely, at later infection time-points when effector T cells are highly activated and progressive reductions in pathogen burden occur, the suppressive potency for Foxp3+ cells is reduced. Together IMP dehydrogenase with the waning impacts of Foxp3+ cell ablation with infection progression, these results illustrate how shifts

in Treg-cell suppression can dictate the tempo of persistent infection.59 Similarly, following acute Listeria infection, reductions in suppressive potency are found for GFP+ Treg cells that immediately precede the expansion of pathogen-specific effector T cells.88 The expansion of circulating Treg cells with increased suppressive potency is associated with increased parasite burdens for patients with severe malaria infection.26 However, no significant changes in suppressive potency were found for Foxp3+ Treg cells isolated directly ex vivo after Plasmodium berghei infection in mice.31 Nevertheless, these findings illustrate how infection-induced shifts in Foxp3+ Treg-cell suppressive potency may play important and increasingly appreciated roles in infection outcomes.

These PRRs can detect a broad range of molecular patterns that are associated with either infection (pathogen-associated molecular pattern; PAMPs) [2, 3] or cell death and trauma (damage-associated molecular patterns; DAMPs) [2, 4]. Following activation through PRRs, DCs undergo a maturation process that is characterized by upregulation of MHC class II and costimulatory molecules on their cell surface, proinflammatory

cytokine production, and DC migration to draining lymph nodes. In the lymph nodes, mature DCs function as the prototype of professional APCs to prime naïve T cells and control T-cell activation [5]. In addition to detecting pathogens or tissue damage directly through PRRs, DCs can be indirectly activated by factors that signal the presence of pathogens. For example, type I interferons (IFNs), which are produced rapidly in the course of viral and bacterial infections, AZD2281 in vivo have been reported to enhance the Ag-presentation R788 cell line efficiency of DCs, as well as DC migration to lymphoid tissues [6]. Moreover, type I IFN receptor signaling in DCs has been found to be essential for

T-cell priming in response to various PAMPs [7], as well as for the induction of virus-specific [8] and tumor-specific T-cell responses [9]. Notably, the interaction of DCs with CD4+ T cells provides additional important stimuli for DC maturation [10]. For example, ligation of CD40 on DCs by CD154 on T cells promotes DC activation, leading to priming of cytotoxic T lymphocytes (CTLs) [11] and CD4+ T-cell differentiation. Over the past decade, it has become clear that, in addition to their role in priming effector T-cell responses against invading pathogens, DCs have a crucial role in self-tolerance. These opposing DC functions are controlled through the regulation of DC maturation in the steady state, and this checkpoint is crucial for the maintenance

of immune homeostasis. In this article, Cell press we review the signals that can induce DC maturation in the steady state and discuss the suppressive mechanisms that counterbalance DC-activating signals to preserve peripheral tolerance. The contribution of steady-state DCs to the maintenance of peripheral tolerance was first shown in animal models, in which Ag could be targeted to immature DCs. Immature steady-state DCs had previously been notoriously difficult to study, as their isolation and manipulation rapidly induce DC maturation [12, 13]. To overcome this problem, the group of Ralph Steinman used mAbs against DC surface receptors to target Ags to DCs in vivo. Antigen delivery to steady-state DCs in the absence of inflammatory signals resulted in a transient activation and proliferation of Ag-specific CD4+ and CD8+ T cells, which was followed by deletion of these T cells and the establishment of Ag-specific T-cell tolerance [14, 15].

Although the IL-10-modulating capacity of Lm clones on LPS-matured DCs described in this study was not strong, it is tempting to speculate that the simultaneous presence of LPS and parasites during leishmaniasis may play a role in the disease progression through an increase of IL-10

production and down-regulation of IL-12. Our results indicate that there is a significant variability in the capacity of Lm clones to infect human DCs. This variability depends upon Lm virulence and could involve LmPDI protein. However, Lm clones modulate find more some signalling pathways favouring their survival in infected DCs independently of their virulence. Furthermore, the capacity of Lm parasites to inhibit CD1a expression strongly may be associated with their capacity to interfere with glycolipid Inhibitor Library concentration presentation, as it has already been demonstrated for L. donovani. Our data present further evidence for the fact that Lm strains can have intrinsic differences in their ability to induce crucial elements of the innate immune response, at least during their initial interactions

with the professional phagocytes. We thank Dr Mehdi Chenik and Sima Drini for manuscript reading (Laboratory of Medical Parasitology, Biotechnology and Biomolecules, Institut Pasteur de Tunis), Dr Narges Bahi-Jaber (Laboratory of Transmission and Immunobiology of Infection, Institut Pasteur de Tunis) for help in statistical analysis, the Blood Transfusion Service of Tunis for blood samples Mannose-binding protein-associated serine protease and especially blood donors for the generous donation of their cells. This work was supported by the Tunisian Ministry for Research and Technology (IMM23).

None. “
“Hepatitis C virus (HCV) has chronically infected an estimated 170 million people worldwide. There are many impediments to the development of an effective vaccine for HCV infection. Dendritic cells (DC) remain the most important antigen-presenting cells for host immune responses, and are capable of either inducing productive immunity or maintaining the state of tolerance to self and non-self antigens. Researchers have recently explored the mechanisms by which DC function is regulated during HCV infection, leading to impaired antiviral T-cell responses and so to persistent viral infection. Recently, DC-based vaccines against HCV have been developed. This review summarizes the current understanding of DC function during HCV infection and explores the prospects of DC-based HCV vaccine. In particular, it describes the biology of DC, the phenotype of DC in HCV-infected patients, the effect of HCV on DC development and function, the studies on new DC-based vaccines against HCV infection, and strategies to improve the efficacy of DC-based vaccines. Hepatitis C virus (HCV) is a blood-borne pathogen and has led to chronic infection in an estimated 170 million people worldwide. It is a major cause of chronic liver diseases with a substantial morbidity and mortality.

40 Recombinant antibodies Selleck MI-503 for clinical therapeutic use in humans are expressed in low yields in mammalian cells, which accounts for their high cost. To cut costs, cPIPP was expressed as a periplasmic protein in tobacco leaves at a high yield of 20 mg of purified protein per Kg fresh tobacco leaves.41 Being given that it was expressed in endoplasmic reticulum of the leaves, plant-specific fucose and xylose residues were not loaded on the antibody.42 cPiPP had an affinity of 1.9 × 1010 m−1 for hCG. It was totally devoid of cross-reaction with hFSH and hTSH and had <5% cross-reaction with hLH. The antibody was fully competent to block hCG-induced gain

of uterine weight of immature mice in vivo and hCG-induced testosterone production by Leydig cells in vitro.40,41 Its efficacy was also tested in a human cell system. Placental villi cytotrophoblasts, isolated from placental villi of MTP cases, on culture in a medium containing anti-hCG antibodies failed to fuse into syncytium. Furthermore, the production of progesterone by the placental cells was fully blocked by cPiPP.26 These observations vouch for the suitability of cPiPP for use as a vacation contraceptive and for non-surgical termination of pregnancy. Choriocarcinoma trophoblast cells are known to make and secrete hCG.43,44 The cells carry receptors for hCG, by virtue of see more which hCG

acts as an autocrine growth factor for these cells. Radio-iodinated PiPP bound to these cells in vitro. JEG cells administered to Nude mice form a cancerous implant. Injection of 131I-PiPP to such mice led to selective localization of radioactivity

at Phosphoglycerate kinase the tumor site, whereas the radioactivity of a similarly radio-iodinated non-relevant antibody is distributed randomly all over the body45 (Fig. 1a,b). The binding of the radio-iodinated PiPP to tumor cells is further confirmed by histioradiography (Fig. 1c). These studies clearly demonstrate the utility of the recombinant antibody for imaging and selective delivery of radiations to the tumor cells. It could be of particular utility for tracing of metastasis of such cancers. The curious phenomenon of cancer cells expressing hCG or its subunits has been discussed elsewhere in this article. We carried out studies on T-lymphoblastic leukemia MOLT-4 and lymphocytic leukemia U-937 cells, both available from ATCC. Both MOLT-4 and U-937 cells were bound with cPiPP. The binding as studied by flow cytometry was on the membranes and was specifically competed by authentic purified hCG.46 hCG was not picked up from other cells but was indeed synthesized by the cancer cells, as permeabilized MOLT-4 cells enabled the detection of the presence of hCG within the cells, to which the antibody permeating in the cells could bind. Incubation of MOLT-4 cells with anti-hCG antibodies did not however impair the viability and multiplication of these cells. Nor were the cells lysed by cPiPP in the presence of complement.

Sciatic nerve was transected, and end-to-end neurorrhaphy was performed on 32 male Sprague-Dawley rats, which were randomly divided into four groups (n = 8 per group): nerve coaptation without treatment (group I); nerve coaptation covered with HA film sheath (group II); nerve coaptation with intramuscular VEGF gene in plasmid injection (group III); and nerve coaptation combined with HA film Anti-infection Compound Library datasheet sheath and intramuscular VEGF gene in plasmid injection (group IV). Contralateral sciatic nerves were used as control. VEGF

expression was verified from gluteal muscle biopsies surrounding the sciatic nerve by reverse transcriptase-PCR. Electrophysiological, histopathological, and electron microscopic evaluations were performed after 4 weeks. Mean peak amplitude of groups I–IV and nonoperated sciatic nerve were 4.5 ± 0.6 mV, 6.4 ± 0.4 mV, 6.7 ± 0.5 mV, 8.5 ± 0.4 mV, and 9.8 ± 0.5 mV, respectively. Mean myelinated axonal counts of groups I–IV and nonoperated sciatic nerve were 105 ± 24, 165 ± 19, 181 ± 22, 271 ± 23, and 344 ± 17, respectively. Treatment with HA film sheath coverage combined with intramuscular VEGF gene in plasmid injection yielded statistically significant

higher peak amplitudes and myelinated axonal counts selleckchem (P < 0.001). In addition, significantly less scar formation with HA administration (groups II and IV; P < 0.001) was found. Thus, it was found that VEGF might crucially regulate nerve regeneration processes and that HA can reduce the scar

formation. This study showed that the combination of HA film sheath and VEGF gene may synergistically promote peripheral nerve regeneration. © 2013 Wiley Periodicals, Inc. Microsurgery 34:209–216, 2014. “
“Venous flow-through flaps are well-described options for Carbohydrate small defects where donor site morbidity is undesirable or in areas where useful local veins are in close proximity to the defect, particularly in the extremities. However, higher rates of flap loss have limited their utility. The saphenous venous flap in particular has been widely sought as a useful flap, and while arterialization of this flap improved survival rates, congestion has remained a limiting feature. We describe report a modification in the design of saphenous venous flaps, whereby an arterialized flap is provided with a separate source of venous drainage, and demonstrate survival of substantially larger venous flaps than previously reported. In five consecutive patients, we describe three main modifications to the saphenous venous flap as previously described: (a) Using arterialized flaps only; (b) Reversing the flap to allow unimpeded flow during arterialization; and (c) Anastomosing additional vein(s) that are not connected to the central vein—especially at the periphery of the flap for true venous drainage.

6b(1)). The selected peptide–H-2Kb interface as the template from crystal structures is presented in Fig. 6b(2).50 NS2:114–121, GQ and FG

peptides are simulated with the same H-2Kb and TCR from the template crystal structure (Fig. 6b(3,4,5)). As the backbones of several H-2Kb-bound peptides adopt the same conformation, we have speculated on many features of the critical contact residues to be the main factors to affect specific recognition by TCR (Figs 6a(2),b). At the fifth anchor motif, substitution of phenylalanine (F) with glycine (G) could undermine the binding forces of GQ to H-2Kb because of the lack of an inward benzyl group without compromising the recognition of the outward side chain via TCR (Fig. 6b(3,4)). The substitution of glutamine (Q) with glycine (G) at the sixth TCR contact site has removed the outward amide side chain find more from recognition by specific TCR (Fig. 6b (3,5)). Simulation results are compatible with those obtained

from laboratory experiments (Tables 2 and 3; Figs 2 and 5). The simulation approach with TCR contact information has more accurate prediction results on epitope identification than all previous computing programmes. Respiratory syncytial virus causes bronchiolitis and pneumonia in infants and young children.51 Influenza A virus still represents one of the major respiratory viruses causing significant morbidity and mortality in severe respiratory tract infections.52 selleck inhibitor In the 1960s, the trials of formalin-inactivated vaccines not only failed to protect those people who were vaccinated from RSV infection but induced deviant pathological consequences.53 The lack of CD8 T-lymphocyte responses has been associated with pulmonary eosinophilia that was observed in vaccinated people or experimental animals.7,53,54 Antigenic drifts and heterotypic influenza A viruses continue to

cause annual epidemics and pandemic outbreaks.4,6 It is critical to identify the important elements constituting the epitope to enable CD8 T-lymphocyte recognition as well as to map mutant epitopes from mutable pathogens, either for experimental research or for immunoinformatical programmes. The role of anchor motifs Thiamet G of peptides in the binding to MHC class I molecules is known and well-studied.19–22 Immunologists and microbiologists have long relied on these anchor motifs to predict MHC class I-restricted epitopes from the protein sequences of viral pathogens. Several peptide–MHC class I binding methods have been developed to map CD8 T-lymphocyte epitopes. Consistent with the previous publication of competitive binding experiments, M2:82–90 had the highest binding affinity to H-2Kd molecules to be detected by RMA-S-Kd cells22 (Figs 1a,c and Supplementary material, Fig. S2).