© 2014 Wiley Periodicals, Inc. Microsurgery, 2014. “
“Heparin-induced

thrombocytopenia AZD0530 in vitro and thrombosis (HITT) is an immune complex mediated and potentially devastating cause of flap loss in microvascular surgery. HITT may be an under-reported cause of early-flap failure due to subclinical manifestations at the time of flap loss. A case report of a patient presenting with HITT-related flap failure and the results of a systematic literature review of the clinical presentation of HITT in microsurgery are presented here. A patient suffering from a chronic wound on the right medial malleolus was treated with an ALT flap, which was compromised by thrombosis. Multiple attempts to rescue the flap including thrombolysis, popliteal AV loop, and a second free flap were all unsuccessful. Six days following the initial procedure, a diagnosis of HITT was made following a positive HITT-antibody test as the cause

of flap failure. PubMed, MEDLINE, and EMBASE searches yielded 113 results, of which 6 met our criteria for manuscripts describing HITT in microsurgical procedures. click here Evaluation of the peer-reviewed literature describing HITT in microsurgery suggests that HITT-related flap failure occurs rapidly, more frequently in heparin-naïve patients, and in advance of systemic thrombosis and thrombocytopenia. Due to the rapid and unpredictable onset of HITT during microsurgery, we recommend maintaining an index of suspicion for HITT in flaps with otherwise unexplained early thrombosis. We also encourage hematology consultation, discontinuing heparin use and initiating alternate thromboprophylaxis in order to inhibit the potential for subsequent life-threatening systemic complications

as well as improving the potential for delayed reconstructive success. © 2013 Wiley Periodicals, Inc. Microsurgery 34:157–163, 2014. “
“Background: Free flaps to the lower limb have inherently high venous pressures, potentially impairing flap viability, which may lead to limb amputation if flap failure ensues. Adequate monitoring of flap perfusion Clomifene is thus essential, with timely detection of flap compromise able to potentiate flap salvage. While clinical monitoring has been popularized, recent use of the implantable Doppler probe has been used with success in other free flap settings. Methods: A comparative study of 40 consecutive patients undergoing microvascular free flap reconstruction of lower limb defects was undertaken, with postoperative monitoring achieved with either clinical monitoring alone or the use of the Cook-Swartz implantable Doppler probe. Results: The use of the implantable Doppler probe was associated with salvage of 2/2 compromised flaps compared to salvage of 2/5 compromised flaps in the group undergoing clinical monitoring alone (salvage rate 100% vs. 40%, P = 0.28).

The values of NS wells were subtracted from those of stimulated wells. The assay was

performed by strictly following the instructions of the BD™ ELISPOT Mouse IFN-γ ELISPOT Selleckchem HSP inhibitor Set (BD, San Diego, CA). Briefly, a 96-well ELISPOT plate was precoated overnight at 4 °C with anti-mouse IFN-γ capture antibody. After one wash with 200 μL per well of blocking solution, 200 μL of blocking solution was added to each well for 2 h at room temperature. The blocking solution was discarded, and a total volume of 100 μL of spleen lymphocyte suspension (adjusted to 2 × 106 cells mL−1) was added to each well. RPMI 1640 medium was supplemented with 10% v/v FBS. The cells were incubated in medium containing 2 μg mL−1 of PPD, 0.8 μg mL−1 of ConA, 16 μg mL−1 of Ag85b, 16 μg mL−1 of HspX, 16 μg mL−1 of C/E or medium alone (no stimulation). After incubation at 37 °C in 5% CO2 for 24 h, cells were removed, MG-132 mouse and the following steps were taken in strict accordance with manufacturer’s instructions. Spots were quantified using

an ELISPOT reader (Cellular Technology Ltd, Shaker Heights, OH). Four percent starch broth (1 mL) was injected into the peritoneum of mice 3 days before sacrifice to yield inflammatory macrophages. After sacrifice, mice were sprayed with 70% alcohol to sterilize the abdomen, then 2 mL of cold Hanks’ balanced salt solution without Ca2+ and Mg2+ (CMF-HBSS) was injected into the peritoneum. After slightly massaging the abdomen for several minutes, the fluid in the peritoneum was collected and centrifuged at 453 g for 10 min. Cells were washed twice with cold CMF-HBSS and then resuspended in Dulbecco’s minimum essential medium (DMEM) (Thermo Scientific) containing 5% FBS. Cells were stained with Diff-Quik staining solution for counting under a microscope. The cell concentration was adjusted to 2.5 × 106 cells mL−1. A 1-mL aliquot of the cell suspension was added to each well of a 24-well plate (Corning) and incubated at 37 °C in 5% CO2 for 2 h. Cells that did not adhere to the wells were discarded, and the wells were washed once with 37 °C DMEM. After the addition of 1 mL of 5% FBS-DMEM to

each well, stimulants were added at the following final concentrations: 10 μg mL−1 of Ag85b, 10 μg mL−1 of HspX and 10 μg mL−1 of LPS. Plates were Bcl-w incubated at 37 °C in 5% CO2 for 48 h, and then culture supernatants were harvested and stored at −80 °C until analysis. IL-12 was determined strictly following the instructions from the Quantikine Mouse IL-12 p70 kit (R&D Systems, Minneapolis, MN). Statistical analysis was performed using graphpad prism version 5.0 for Windows (GraphPad Software, San Diego, CA). Data analyses for antibody response, lymphocyte proliferation and concentration of IL-12 were performed using a one-way anova on the raw data, and the analyses for ELISPOT, total lesion scores and bacterial load results were performed using the rank sum test.

2A). Confirming results obtained on total NK cells, expression of KIR2DL1 but not of KIR3DL1 increased on NKG2C+ cells (Fig. 2C and D). Interestingly, a small but statistically significant increase in KIR2DL1 on NKG2C+ was detected also in CMV-seronegative donors; however, this increase was much smaller than that seen in selleck chemicals CMV-seropositive donors (Fig. 2C). To discriminate between expression of KIR2DL2/S2 and KIR2DL3, we next cultured PBMCs from donors carrying

the genes for all three receptors. Co-staining of a KIR2DL3 specific Ab with an Ab recognizing KIR2DL2/S2/L3 allowed us to distinguish between expression of KIR2DL2/S2 and KIR2DL3 (Fig. 3A). In five CMV-seropositive donors, strong expansion of KIR2DL3-expressing NK cells was documented, while co-culture with

CMV-infected fibroblasts had no impact on the expression of KIR2DL2/S2 (Fig. 3B and C). To address whether the increased expression of KIR-expressing cells represents true expansion, we determined cell number weekly during the 21-day co-culture with MRC-5 in the presence or absence of CMV. The RG7204 manufacturer NK-cell number contracted during the first week, followed by an expansion of NK cells exclusively in seropositive donors in the presence of CMV (Supporting Information Fig. 2). Staining for the proliferation marker Ki-67 corroborated these results: infection of MRC-5 with CMV led to a massive up-regulation of Ki-67 on NK cells if these stemmed from CMV-seropositive donors (Fig. 2B). Interestingly, when the KIR repertoire was assessed on Ki-67+ cells, we noted expansion of KIR2DL1/Ki-67 double positive but not of KIR3DL1/Ki-67 double positive cells after co-culture with CMV-infected MRC-5 (Fig. 2E and F). We next aimed

to characterize factors www.selleck.co.jp/products/lee011.html influencing the expansion of KIR-expressing NK cells. HLA-C1 group Ags are the ligand for KIR2DL2/S2/L3, while HLA-C2 group Ags are the ligands to KIR2DL1 [17]. If CMV-seropositive donors were stratified according to their KIR ligand status, an expansion of KIR2D-expressing NK cells occurred only in the presence of the cognate KIR ligand: KIR2DL1 expanded only in donors carrying a C2 ligand (Fig. 4A and B), whereas KIR2DL2/S2/L3 NK cells expanded exclusively in the presence of the cognate group C1 ligand (Fig. 4C and D). While no ligand has been identified for the activating KIR receptor KIR3DS1 [18], genetic association studies have suggested an epistatic interaction of KIR3DS1 with HLA-Bw4 in HIV infection [19]. Analysis of Bw4-status in conjunction with KIR3DS1 expression in our population showed that expansion of KIR3DS1 occurred irrespective of the presence of Bw4 (day 21 KIRDS1 expression in CMV-exposed versus CMV nonexposed cells in seropositive donors: mean 23 versus 8% in Bw4-negative, and 31 versus 11% in Bw4-positive donors, p < 0.05 for both comparisons).

Furthermore, the efficiency whereby Treg cells silence immune activation coupled with the plasticity in Foxp3+ cell activity suggest that 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+ Treg cells in host defence against infection, and the significance of infection-induced shifts in Treg-cell suppression are summarized. The fluid balance between immune activation required for optimal host defence against Palbociclib price infection and immune suppression that maintains tolerance by averting autoimmunity is stringently regulated. This allows immune effectors with CB-839 purchase the potential to cause catastrophic damage to host tissues to be actively silenced during homeostasis, but also rapidly unleashed in response to infection. Accordingly, the cell-associated and cytokine signals that stimulate the activation of immune effectors have been intensely investigated for developing new therapeutic strategies

for boosting desired immune responses during infection or immunization. On the other hand, understanding how ubiquitous immune suppression signals are selectively silenced during immune activation, and the extent to which they limit optimal host defence against infection has lagged behind. This bottleneck has been overcome with the identification of a distinct

CD4+ T-cell subset with immune suppressive properties called regulatory T (Treg) cells.1–3 Although Treg cells were initially identified as the CD4+ T-cell subset that constitutively express the interleukin-2 (IL-2) receptor, CD25, subsequent landmark studies have since established that the lineage-defining and master regulator for Treg cells is dictated by expression of the forkhead box P3 transcription factor, Foxp3.4–6 Infants who develop oxyclozanide a fatal rare constellation of clinical features that includes refractory eczema, diabetes, thyroiditis, colitis, infection susceptibility and generalized wasting called the immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome have mutations in either the foxp3 promoter or coding sequence resulting in defective Treg cells.7–9 Similarly, mice with naturally occurring or targeted defects in foxp3 develop similar clinical features (lymphoproliferation, colitis, weight loss, diabetes and ruffled hair) associated with systemic autoimmunity, and become moribund within 20–25 days of birth.6,8,10 Accordingly, Foxp3+ Treg cells are essential for maintaining peripheral immune tolerance in humans and mice, and these parallels in clinical features with Treg deficiency illustrate the usefulness of mouse models to investigate how Treg cells may control other facets of the immune response.

Detailed descriptions of all individuals are shown in Table 1. Collection and storage of serum samples.  Blood samples were collected before any treatment initiation. The whole blood samples were collected in 4 ml BD Vacutainers without anticoagulation and clotted at room temperature for up to 1 h, and then samples were centrifuged at 4 °C for 5 min at 9000 g. Immediately, collected, aliquoted and stored these fresh sera at −80 °C to avoid variations

in the procedure. No sample underwent more than one freeze-thaw cycle before analysis. Serum pretreatments and MALDI-TOF MS detection.  Serum samples were pretreated with WCX magnetic beads of protein fingerprinting detection kit (SED™) (Beijing SED Science and Technology, Inc., Beijing, China). Briefly, 5 μl of each serum sample was mixed with 10 μl of U9 solution in a 0.5 μl centrifuge Copanlisib cost tube for denaturation. After incubating for 30 min at room temperature, denatured serum sample was diluted with 185 μl washing buffer. Meanwhile, 50 μl of magnetic beads was added to a PCR tube, and the tube was placed in a magnet separator for 1 min followed by carefully removing the supernatant. The magnetic beads were then washed twice with 100 μl washing buffer. Hundred microlitre of diluted serum sample was added to the activated

magnetic beads, mixed carefully and thoroughly. The mixture was incubated for 1 h at room temperature and then washed twice with 100 μl washing buffer. The bound proteins were eluted from the magnetic beads

using 10 μl INCB024360 elution buffer. Then, 4 μl of the eluted sample was diluted in the ratio of 1:2 with 4 μl of SPA (saturated solution of sinapinic acid in 50% acetonitrile with 0.5% trifluoroacetic acid). Two microlitre of clonidine the resulting mixture was aspirated and spotted onto an 8-spot Au-chip (Ciphergen Biosystems Inc., Fremont, CA, USA). After air-drying for about 5 min at room temperature, protein crystals on the chip were detected by MALDI-TOF MS (Ciphergen, PBS IIc). The instrument was calibrated weekly using the Ciphergen all-in-one peptide reference standard, which contained vasopressin (1084 Da), somatostatin (1637 Da), bovine insulin β chain (3495 Da), human insulin recombinant (5807 Da), hirudin (7033 Da). And mass calibration helps guarantee that mass error was <3 Da. The detective parameters of MALDI-TOF MS were as follows: optimized mass range (2000–20,000 Da), laser intensity (149), laser sensitivity (7). It started with two warming shots at intensity of 154, then 110 shots at laser intensity of 149. Eighty-eight shots of the latter set were randomly kept, and results were generated from their average level. All the information including mass and intensity of peaks over the range mass/charge ratio (m/z) 0–50,000 Da was collected by ProteinChip@ Software Version 3.21 (PCS; Ciphergen). Data processing.  Spectra from all samples were initially processed with baseline subtraction and normalization using PCS.

After 24 h, cells were transduced with retroviral supernatant by spin-infection 49 and cultured for a further 3–4 days before transferring sorted eGFP+ BM cells into recipient mice preconditioned with 2×550 cGy total body irradiation.

Between 20 000 and 200 000 eGFP+ cells were transferred via tail intravenous injections. One day later, radioresistant host T cells were depleted by treatment of BM recipients and untreated control groups with anti-Th1.1 (clone T24) antibody. Mice were left to Selleck Saracatinib reconstitute for 8–10 weeks before immunisation. Levels of chimerism were determined 5 weeks post BMT through blood analysis and extensively at completion of experiment. mTEC were enriched from thymus as described by Gray et al. 51. Thymi from 10–12 adult mice (6–10 weeks old) were collected in MT-RPMI.

After the removal of excess fat and connective tissue, small cuts were made around the edges of the thymic lobes. Following a brief agitation using a wide bore glass pipette, the sample was then subjected to enzymatic digestion. Thymic fragments were incubated in 5 mL of 0.125% w/v collagenase D with 0.1% w/v DNAse I (Roche) in MT-RPMI at 37°C for 15 min. Cells released into suspension were removed after larger thymic fragments had settled and fresh enzyme containing media was added to the intact thymic lobes. This was repeated 3–4 times with fresh media. In the final digest, collagenase D was replaced with trypsin (Roche) and incubation time was extended to allow for complete digestion of thymi lobules. Each fraction was counted and the final 2 find more or 3 enrichments, which contained a higher proportion this website of CD45– cells, were pooled to obtain 100×106 total cells. A negative depletion was performed to enrich for CD45– cells using CD45 microbeads (Miltenyi Biotec) and the AutoMACS system (Miltenyi Biotec), using the DepleteS program. The CD45– cell fraction was then resuspended in KDS-BSS with 3% v/v FBS and stained using the following antibodies: anti-CD45-APC (30F11; BD Biosciences), anti-MHCII-PE (M5/114.15.2; BD Biosciences) and anti-Ly51-FITC (6C3; BD Biosciences).

Prior to sorting, 0.5 μg/mL PI (Calbiochem) was added to each samples to allow for the exclusion of dead cells. Cells were sorted using the FACSAria (BD Biosciences). RNA from cultured cells, whole tissues or sorted cells was prepared using the RNeasy Mini-kit (Qiagen) including an on-column DNaseI digest as per manufacturer’s protocol. cDNA was generated using Superscript III RT (Invitrogen) as per manufacturer’s protocol. For RT-PCR the primers used were: Aire; For 5′-accatggcagcttctgtccag-3′, Rev 5′-gcagcaggagcatctccagag-3′; Ins2; For 5′-accatcagcaagcaggaag-3′, Rev 5′-ctggtgcagcactgatctacaatgc-3′; Mog; For 5′-ggactagtgactctgtccccggtaaccat-3′, Rev 5′-ggactagtctcgagagaaccatcactcaaaagggg-3′, Gapdh; For 5′-catgacaactttggcattgtgg-3′, Rev 5′-cagatccacaacggatacattggc-3′. PCR conditions were optimized for each primer set.

In the same blood monocytes, the secretion of IL-18 following LPS stimulation is consistently low and, compared with IL-1β, negligible. By comparison, IL-1β is readily released following LPS stimulation in the absence of added

ATP because caspase-1 is already active in fresh monocytes [[8]]. In contrast, Epigenetics inhibitor macrophages require activation of caspase-1 with substantial concentrations of ATP [[8]]. Thus, the robust release of processed IL-1β compared with the weak release of processed IL-18 reveals that the mechanism of release from the postcaspase-1 cleavage step is not the same for these two cytokines. Indeed, a lingering question is why this difference exists. One possible explanation is that the constitutive presence of the IL-18 precursor in monocytes remains in the cytoplasm whereas the newly synthesized Ku-0059436 mouse IL-1β precursor enters the secretory lysosome where it is processed by caspase-1 and exported [[9, 10]]. With the report by Bellora et al. in this issue of the European Journal of Immunology [[11]], the similarity of IL-18 to IL-1α now becomes closer with the observation that a membrane form of IL-18 is found on a subset of monocyte-derived macrophages following exposure to macrophage colony-stimulating factor (M-CSF). Similar to IL-1α, membrane IL-18 is an active cytokine only upon stimulation with TLR ligands such as

LPS [[12, 13]]. This is an important similarity for IL-1α and IL-18 in that LPS stimulation triggers a step resulting in an active cytokine. Membrane cytokines are not new to cytokine biology. TNF-α can exist in a membrane form, and requires a protease for release. However, the

first report of a functional membrane cytokine was that of IL-1α in 1985 [[12]]. This milestone was at first appreciated for its relevance to the biology of the IL-1 family, then questioned and finally resolved. The insertion of IL-1α into the membrane is possible because of myristoylation of the IL-1α precursor at lysines 82 and 83, a step that facilitates the insertion into the membrane [[14]]. There is Casein kinase 1 a potential myristoylation site in the IL-18 precursor but it remains unclear if this site accounts for insertion into the membrane. There are unique findings in the study by Bellora et al. [[11]]. First, the appearance of membrane IL-18 is slow given the fact that the monocyte already contains the precursor. Second, its appearance is linked to the differentiation into an M2-type macrophage by exposure to M-CSF whereas differentiation into an M1-type macrophage by exposure to GM-CSF does not result in membrane IL-18. Third, although its presence on the membrane of the differentiated M2 macrophage is caspase-1 dependent, the cytokine is inactive. Activation requires LPS.

These discussions provided the basis of the contents of this manuscript. The following sections summarize the opinions and recommendations on clinical practice and future research directions. These categories, characterized by mesangial immune deposits with or without mesangial proliferation under light microscopy, are often not accompanied by acute nephritic syndrome or heavy proteinuria. The KDIGO guideline recommends that management should be based on concomitant extra-renal lupus manifestations if present.[16] Nephrotic syndrome due to concomitant podocytopathy would warrant treatment with corticosteroids. The majority of patients respond to high-dose corticosteroids, but the addition

of an immunosuppressive AZD2014 research buy agent may be necessary when response is unsatisfactory and in frequent relapsers. Low-to-moderate doses of prednisone (0.25–0.5 mg/kg/day) alone or in combination with azathioprine is recommended by the EULAR guidelines for Class

II LN with proteinuria >1 g/24 hr despite renin-angiotensin-aldosterone system blockade.[17] The natural course of severe proliferative LN is progressive immune-mediated inflammation and destruction of nephrons, although the severity and rate of progression vary widely between individuals. Prompt ablation of disease activity and inflammatory damage to nephrons is of critical importance. Delay of treatment, even if effective, results in reduced renal reserve and increases the risk of chronic renal failure. Both the KDIGO and ACR guidelines recommend initial LY2835219 mouse very therapy with high-dose corticosteroids in combination with either CYC or MMF for Class III or IV LN (Table 2).[16,

18] The KDIGO guidelines recommend a change to alternative therapy or a repeat kidney biopsy for assessment in patients who show worsening disease during the first three months of treatment, while the ACR guidelines suggest the decision to change treatment be made at six months.[16, 18] There is considerable variation in the corticosteroid dosage regimen in different guidelines, and the regimens have not been compared in controlled trials. Intravenous pulse methylprednisolone for 3 days followed by oral prednisolone (0.5–1.0 mg/kg/day for a few weeks, then tapered to lowest effective dose) is recommended by ACR,[18] based on results of previous studies[8, 9, 19] and the objective of avoiding excessive cumulative exposure to corticosteroids. When pulse methylprednisolone is not used, all the three guidelines recommend a higher initial dose of oral prednisone (up to 1.0 mg/kg/day), especially when there is histological evidence of aggressive disease such as the presence of any crescents.[16-18] Effective in Whites, Blacks and Chinese Easy to administer and lower cost than oral CYC An extended course of CYC (30 months), compared to shorter courses of approximately 6 months, was associated with fewer renal relapses but more toxicities such as cervical intra-epithelial neoplasia.

) for determination of the flanking

regions of the insertion. Genomic DNA of mutants were prepared as described above. The first PCR reaction was performed with eight different primer pairs in which one of the DW-ACPs was combined with EZTN-F or EZTN-R. PCR amplification was carried out at 94 °C for 5 min, 42 °C for 1 min, 72 °C for 2 min, and then 30 cycles of 94 °C for 40 s, 55 °C for 40 s, and 72 °C for 1 min, followed by 72 °C for 7 min. The first nested PCR was performed using primer pairs of EZ-Tn5 Tnp-specific nested primers KAN2-1or KAN2-3R (Table 1) and a DW-ACP for nested PCR (DW-ACPN: AZD5363 nmr 5′-ACPN-GGTC-3′) provided by the kit (Seegene Inc.). Two microliters of the first PCR product was used as template DNA. PCR amplification was carried out at 94 °C for 5 min, and then 35 cycles of 94 °C for 40 s, 60 °C for 40 s, and 72 °C for 1 min, followed by 72 °C for 7 min. The second

nested PCR was performed using Tofacitinib clinical trial primer pairs of EZ-Tn5 Tnp-specific second nested primers (KAN-2FP1 or KAN-2RP1 provided by the EZ-Tn5 Tnp Kit (Epicentre Biotechnologies, Table 1) and a universal primer (5′-TCACAGAAGTATGCCAAGCGA-3′) provided by the kit (Seegene Inc.). One microliter of the first nested PCR product was used as template DNA. Conditions for PCR were as follows: 94 °C for 5 min, then 35 cycles at 94 °C for 40 s, 60 °C for 40 s, and 72 °C for 1 min, followed by 72 °C for 7 min. The PCR products were electrophoresed, isolated, and cloned using the TOPO TA Cloning system (Invitrogen). Plasmids containing the

PCR products were purified using the QIAprep Spin MiniPrep Kit (Qiagen Science, MD). The PCR products were then sequenced using the Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, Foster City, CA) with a pair of M13 primers. The DNA sequences obtained were converted into amino acid sequences using genetyx ver. 7.0 software (Genetyx www.selleck.co.jp/products/pazopanib.html Co. Ltd, Tokyo, Japan). Homology searches of amino acid sequences were performed using the fasta algorithm in the DDBJ (Mishima, Japan). The sequence of the flanking regions of the EZ-Tn5 Tnp insertion has been submitted to the DDBJ nucleotide sequence database (DDBJ accession: AB377402). Among 486 mutants, we found only one mutant (strain 455) that had lost the ability to produce exopolysaccharide and form meshwork-like structures. The sequencing analysis of the flanking regions of the transposon insertion revealed that the transposon was inserted into an ORF highly homologous to wzt in the per cluster of Y. enterocolitica serotype O:9 (Lubeck et al., 2003; Skurnik, 2003; Jacobsen et al., 2005).

Thus, DNGR-1 targeting allows for MHC class II presentation by CD8α+ DC in vivo. MHC class II:peptide complexes generated after targeting to CD8α+ DC using DEC205-specific mAb are not stable with time 21. To test whether the same was true when anti-DNGR-1 mAb was used as vector, we injected B6 mice with OVA323–339-coupled anti-DNGR1 mAb and analyzed MHC class II presentation by DC at different

time points. Consistent with the kinetics of in vivo staining, CD8α+ but not Z-VAD-FMK molecular weight CD8α− DC were able to efficiently present antigen to OT-II cells as early as 1 h after injection (Fig. 1C). Antigen presentation peaked at 6 h but was markedly reduced by 24 h (Fig. 1C). Thus, antigen targeting to CD8α+ DC using anti-DNGR-1 mAb in the absence of adjuvant leads to rapid but short-lived antigen presentation on MHC class II molecules. To monitor presentation directly in vivo, we transferred CFSE-labeled OT-II cells and 1 day later, we injected the mice with 0.5 μg of OVA323–339-coupled anti-DNGR-1 mAb, 5 μg of OVA323–339-coupled isotype-matched control, 20 μg of OVA (in the form of egg white 22; OVAegg) or 1 μg of OVA323–339 peptide. Administering antigen in untargeted form led only to limited proliferation of OT-II cells, while targeting to DNGR-1

resulted in marked cell division and accumulation (Fig. 2A). On a molar basis, we estimate that targeting to DNGR-1 was 10 to 100 times more efficient at inducing CD4+ T-cell expansion than delivery of untargeted antigen. Thus, despite the restriction of presentation to a short period of time following antigen delivery check details (Fig. 1C), DNGR-1 targeting can induce CD4+ T-cell proliferation in

vivo, as recently reported 17. Injection of anti-DNGR-1 mAb did not lead to any detectable activation of splenic CD8α+ DC (not shown). Nevertheless, we evaluated whether antigen targeting to DNGR-1 could lead to CD4+ T-cell priming in the absence of adjuvant, as recently suggested 17. To avoid any contribution from memory or Treg, we transferred sorted naïve OT-II lymphocytes into B6 mice. One day later, the mice were injected with 0.5 μg of OVA323–339-coupled anti-DNGR-1 mAb with or without 40 μg of poly I:C, a TLR3 and RIG-I/MDA5 agonist recently described as the most potent Th1-promoting adjuvant in experiments of antigen targeting to DEC205 23. In GNAT2 the absence of poly I:C, we observed CD4+ T-cell expansion but no detectable differentiation into Th1, Th2 or Th17 cells (Fig. 2B and C and data not shown). Consistent with the absence of immunity in these conditions, the mice did not develop a strong Ab response to rat IgG following anti-DNGR-1 injection (Fig. 3A). Low titers of anti-rat antibodies were detected only when injecting a higher dose of anti-DNGR-1 mAb (Fig. 3C), matching the one used in a previous report 17. However, the anti-rat IgG response seen with anti-DNGR-1 alone was dwarfed by that which could be induced by co-administration of poly I:C (Fig. 3C).