InVivoMAb anti-mouse MHC Class I (H-2Kb) bound to SIINFEKL peptide (OVA residues 257-264)
Product Details
The 25-D1.16 monoclonal antibody reacts with mouse MHC class I H-2Kb bound to the ovalbumin-derived peptide with sequence SIINFEKL. This antibody does not react with unbound MHC class I H-2Kb or MHC class I H-2Kb bound to an irrelevant peptide. The 25-D1.16 antibody is often used to track the quantity and localization of antigen-presenting cells bearing these specific molecules in vivo.Specifications
| Isotype | Mouse IgG1, Īŗ |
|---|---|
| Recommended Isotype Control(s) | InVivoMAb mouse IgG1 isotype control, unknown specificity |
| Recommended Dilution Buffer | InVivoPure pH 7.0 Dilution Buffer |
| Immunogen | SIINFEKL pulsed RMA-S cells |
| Reported Applications |
in vivo blocking of Kb -SIINFEKL Functional assays Flow cytometry |
| Formulation |
PBS, pH 7.0 Contains no stabilizers or preservatives |
| Endotoxin |
<2EU/mg (<0.002EU/Ī¼g) Determined by LAL gel clotting assay |
| Sterility | 0.2 Ī¼M filtered |
| Production | Purified from tissue culture supernatant in an animal free facility |
| Purification | Protein G |
| RRID | AB_10950697 |
| Molecular Weight | 150 kDa |
| Storage | The antibody solution should be stored at the stock concentration at 4Ā°C. Do not freeze. |
Recommended Products
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Recommended Isotype Control(s)
InVivoMAb mouse IgG1 isotype control, unknown specificity
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Recommended Dilution Buffer
InVivoPure pH 7.0 Dilution Buffer
Functional assays
CD8(+) T cell activation by murine erythroblasts infected with malaria parasites PubMed
Recent studies show that some human malaria parasite species Plasmodium falciparum and P. vivax parasitize erythroblasts; however, the biological and clinical significance of this is unclear. To investigate further, we generated a rodent malaria parasite (P. yoelii 17XNL) expressing GFP-ovalbumin (OVA). Its infectivity to erythroblasts was confirmed, and parasitized erythroblasts were capable of initiating malaria infections. Experiments showed that MHC class I molecules were highly expressed on parasitized erythroblasts. As CD8(+) T cells recognize MHC class I and peptide complexes on target cells, and are involved in protection or pathology against malaria, we examined whether erythroblasts are targeted by CD8(+) T cells. Purified non-parasitized erythroblasts pulsed with OVA peptides were recognized by OVA-specific CD8(+) T cells. Crucially, parasitized erythroblasts isolated from GFP-OVA-, but not GFP- infected-mice, activated OT-I CD8(+) T cells, indicating that CD8(+) T cells recognize parasitized erythroblasts in an antigen-specific manner.
Flow Cytometry
CD4 T-cells transduced with CD80 and 4-1BBL mRNA induce long-term CD8 T-cell responses resulting in potent antitumor effects PubMed
Therapeutic cancer vaccines are an attractive alternative to conventional therapies to treat malignant tumors, and more importantly, to prevent recurrence after primary therapy. However, the availability of professional antigen-presenting cells (APCs) has been restricted by difficulties encountered in obtaining sufficient professional APCs for clinical use. We have prepared an alternative cellular vaccine with CD4 T-cells that can be expanded easily to yield a pure and homogeneous population in vitro. To enhance their potency as a therapeutic vaccine, in vitro expanded CD4 T-cells were transfected with RNAs encoding the costimulatory ligands CD80, 4-1BBL, or both (CD80-T, 4-1BBL-T, and CD80/4-1BBL-T-cells, respectively). We observed augmented cell vitality in CD80/4-1BBL-T-cells in vitro and in vivo. Significant CD8 T-cell responses eliciting in vivo proliferation and cytotoxicity were obtained with CD80/4-1BBL-T-cell vaccination compared to CD80-T and 4-1BBL-T-cell vaccinations. In contrast, beta2m-deficient CD80/4-1BBL-T-cells were not as effective as wile-type CD80/4-1BBL-T-cells in priming CD8 T-cells. Furthermore, CD80/4-1BBL-T-cell immunization resulted in curing established EG7 tumors, resulting in the generation of memory CD8 T-cell responses, and elicited therapeutic antitumor responses against B16 melanoma. These results suggest that CD4 T-cells endowed with costimulatory ligands allow the design of effective vaccination strategies against cancer.
Flow Cytometry
Thymoproteasomes produce unique peptide motifs for positive selection of CD8(+) T cells PubMed
Positive selection in the thymus provides low-affinity T-cell receptor (TCR) engagement to support the development of potentially useful self-major histocompatibility complex class I (MHC-I)-restricted T cells. Optimal positive selection of CD8(+) T cells requires cortical thymic epithelial cells that express beta5t-containing thymoproteasomes (tCPs). However, how tCPs govern positive selection is unclear. Here we show that the tCPs produce unique cleavage motifs in digested peptides and in MHC-I-associated peptides. Interestingly, MHC-I-associated peptides carrying these tCP-dependent motifs are enriched with low-affinity TCR ligands that efficiently induce the positive selection of functionally competent CD8(+) T cells in antigen-specific TCR-transgenic models. These results suggest that tCPs contribute to the positive selection of CD8(+) T cells by preferentially producing low-affinity TCR ligand peptides.
Flow Cytometry
The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells PubMed
The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER.
Flow Cytometry
Peptide-MHC-I from Endogenous Antigen Outnumber Those from Exogenous Antigen, Irrespective of APC Phenotype or Activation PubMed
Naive anti-viral CD8+ T cells (TCD8+) are activated by the presence of peptide-MHC Class I complexes (pMHC-I) on the surface of professional antigen presenting cells (pAPC). Increasing the number of pMHC-I in vivo can increase the number of responding TCD8+. Antigen can be presented directly or indirectly (cross presentation) from virus-infected and uninfected cells, respectively. Here we determined the relative importance of these two antigen presenting pathways in mousepox, a natural disease of the mouse caused by the poxvirus, ectromelia (ECTV). We demonstrated that ECTV infected several pAPC types (macrophages, B cells, and dendritic cells (DC), including DC subsets), which directly presented pMHC-I to naive TCD8+ with similar efficiencies in vitro. We also provided evidence that these same cell-types presented antigen in vivo, as they form contacts with antigen-specific TCD8+. Importantly, the number of pMHC-I on infected pAPC (direct presentation) vastly outnumbered those on uninfected cells (cross presentation), where presentation only occurred in a specialized subset of DC. In addition, prior maturation of DC failed to enhance antigen presentation, but markedly inhibited ECTV infection of DC. These results suggest that direct antigen presentation is the dominant pathway in mice during mousepox. In a broader context, these findings indicate that if a virus infects a pAPC then the presentation by that cell is likely to dominate over cross presentation as the most effective mode of generating large quantities of pMHC-I is on the surface of pAPC that endogenously express antigens. Recent trends in vaccine design have focused upon the introduction of exogenous antigens into the MHC Class I processing pathway (cross presentation) in specific pAPC populations. However, use of a pantropic viral vector that targets pAPC to express antigen endogenously likely represents a more effective vaccine strategy than the targeting of exogenous antigen to a limiting pAPC subpopulation.
Flow Cytometry
Dipeptides catalyze rapid peptide exchange on MHC class I molecules PubMed
Peptide ligand selection by MHC class I molecules, which occurs by iterative optimization, is the centerpiece of immunodominance in antiviral and antitumor immune responses. For its understanding, the molecular mechanisms of peptide binding and dissociation by class I molecules must be elucidated. To this end, we have investigated dipeptides that bind to the F pocket of class I molecules. We find that they accelerate the dissociation of prebound peptides of both low and high affinity, suggesting a mechanism of action for the peptide-exchange chaperone tapasin. Peptide exchange on class I molecules also has practical uses in epitope discovery and T-cell monitoring.
in vivo blocking of Kb -SIINFEKL
Interstitial Migration of CD8Ī±Ī² T Cells in the Small Intestine Is Dynamic and Is Dictated by Environmental Cues PubMed
The migratory capacity of adaptive CD8Ī±Ī² T cells dictates their ability to locate target cells and exert cytotoxicity, which is the basis of immune surveillance for the containment of microbes and disease. The small intestine (SI) is the largest mucosal surface and is a primary site of pathogen entrance. Using two-photon laser scanning microscopy, we found that motility of antigen (Ag)-specific CD8Ī±Ī² T cells in the SI is dynamic and varies with the environmental milieu. Pathogen-specific CD8Ī±Ī² T cell movement differed throughout infection, becoming locally confined at memory. Motility was not dependent on CD103 but was influenced by micro-anatomical locations within the SI and by inflammation. CD8 T cells responding to self-protein were initially affected by the presence of self-Ag, but this was altered after complete tolerance induction. These studies identify multiple factors that affect CD8Ī±Ī² T cell movement in the intestinal mucosa and show the adaptability of CD8Ī±Ī² T cell motility.
