Assessments of Roles for Calreticulin in Antigen Presentation.

MHC class I molecules are important glycoproteins of the adaptive immune response. Assembly of MHC class I molecules occurs in the endoplasmic reticulum (ER) of cells. A group of proteins termed the MHC class I peptide-loading-complex (PLC) facilitate the assembly of MHC class I molecules. Calreticulin, a member of the PLC, is a soluble ER chaperone that aids in the folding of nascent glycoproteins via a glycan-binding site contained within a globular lectin-like domain. Calreticulin also interacts with ERp57, a thiol-oxidoreductase that facilitates disulfide isomerization in calreticulin-associated glycoproteins. Although it is widely accepted that calreticulin utilizes its lectin-like domain to chaperone glycoproteins, calreticulin can also suppress aggregation of non-glycosylated substrates in vitro under conditions of ER-stress. Thus, the cellular modes of substrate binding by calreticulin remain unclear and somewhat controversial. To this end, we focus on characterizing requirements for calreticulin recruitment into the PLC and identifying substrates of calreticulin within the PLC. Although calreticulin is typically ER-localized, under conditions of cell-stress, tumorigenesis, or cell-death, calreticulin migrates to the cell-surface where it acts as a pro-phagocytic signal. Soluble extracellular calreticulin is also suggested to mediate endocytosis of associated antigen. Thus, we investigated whether calreticulin-mediated phagocytosis or endocytosis translates to enhanced presentation of exogenous antigen to CD8 T-cells. In sum, we assess mechanisms relevant to calreticulin-mediated protein folding in the ER, and impacts of extracellular calreticulin upon presentation of exogenous antigen to CD8 T-cells. We show that calreticulin recruitment into the PLC is mediated via glycan and ERp57-dependent interactions. Additionally tapasin, an assembly factor for MHC class I molecules, is a key substrate for calreticulin recruitment into the PLC, and tapasin itself is a calreticulin substrate. We also show that the pro-phagocytic/endocytic role of calreticulin per se does not impact CD8 T-cell responses against cell-associated, peptide, bead-associated, or fused antigen. We investigate factors that may have rendered calreticulin-mediated phagocytosis non-essential, including impacts of innate stimulation and alternative modes of antigen transfer. Together, these findings allow for a better understanding of the chaperone function of calreticulin in the ER and mechanisms relevant to inducing CD8 T-cell responses against soluble and cell-associated antigens.PHDImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/94105/1/ndelcid_1.pd

Assessment of roles for calreticulin in the cross-presentation of soluble and bead-associated antigens.

Antigen cross-presentation involves the uptake and processing of exogenously derived antigens and their assembly with major histocompatibility complex (MHC) class I molecules. Antigen presenting cells (APC) load peptides derived from the exogenous antigens onto MHC class I molecules for presentation to CD8 T cells. Calreticulin has been suggested to mediate and enhance antigen cross-presentation of soluble and cell-derived antigens. In this study, we examined roles for calreticulin in cross-presentation of ovalbumin using a number of models. Our findings indicate that calreticulin does not enhance in vitro cross-presentation of an ovalbumin-derived peptide, or of fused or bead-associated ovalbumin. Additionally, in vivo, calreticulin fusion or co-conjugation does not enhance the efficiency of CD8 T cell activation by soluble or bead-associated ovalbumin either in wild type mice or in mice lacking Toll-like receptor 4 (TLR4). Furthermore, we detect no significant differences in cross-presentation efficiencies of glycosylated vs. non-glycosylated forms of ovalbumin. Together, these results point to the redundancies in pathways for uptake of soluble and bead-associated antigens

Cross-presentation of a peptide antigen.

<p>(A) 10 µM calreticulin (CRT) or bovine serum albumin (BSA) were incubated with 10 µM peptide (QLESIINFEKLTE-FITC). Free peptide was removed using a centrifugal filter device at 4°C, and peptide still in complex with CRT or BSA was measured (left panel). CRT- or BSA-peptide complexes were incubated with BMDC and B3Z cells. IL-2 production was determined by ELISA of the supernatants after 24 hours (right). Peptide concentration is indicated; CRT or BSA were present at a final concentration of 1 µM. (B) Cross-presentation of free peptide or CRT-peptide complexes was measured as in <i>A</i>. Data are representative of two independent analyses for both <i>A</i> (right panel) and <i>B</i>. Mean ± s.e.m. are shown in A and B.</p

In vivo cross-presentation of glycosylated and non-glycosylated OVA.

<p>WT recipient mice were injected i.v. with CFSE labeled OT-I T cells. Twenty-four hours later, mice received s.c. injections of the indicated antigen (2.5–100 µg). OT-I T cell proliferation was measured 3–5 days later in the dLN (inguinal). Left panel: A representative proliferation profile is presented in response to 2.5 µg OVA. Middle and right panels: Two to three mice are averaged to generate each data point, which represents the % of proliferating OT-I T cells (middle) or the % of OT-I T cells as a function of all CD8 T cells recovered (right). Three independent experiments are represented. In one experiment, 2 different OVA (<i>E. coli</i>) preps were used in 2 different groups of mice. In another experiment, two doses (10 µg and 100 µg) of OVA were used in 2 different groups of mice. A two-tailed pair-wise student t-test was used for statistical analysis.</p

<i>In vivo</i> cross-presentation of a calreticulin-fused soluble antigen.

<p>(A, B) WT or TLR4^−/− recipient mice were injected i.v. with CFSE labeled OT-I T cells. Twenty-four hours later, mice received s.c. injections of the indicated antigen (100 µl of a 220 nM solution). OT-I T cell proliferation was measured 3 days later in the dLN (inguinal). (A) The % of proliferating OT-I T cells averaged from the mice of one experiment is shown in the left panel. Two to three mice were used in all groups. The right panel depicts proliferation in WT recipient mice. (B) Quantification of the % of OT-I T cells of all CD8 T cells recovered in <i>A</i>. Data for <i>A</i> and <i>B</i> are representative of three out of four independent analyses for WT recipients and a single analysis with TLR4^−/− recipients. Similar results were obtained in comparisons of OVA and OVA-CRT-induced OT-I proliferation in WT and TLR2/4^−/− recipient mice (data not shown). (C) Compilation of the % of proliferating OT-I T cells (left panel) and of the % of OT-I T cells as a function of all CD8 T cells (right panel) from 4 independent experiments performed with WT recipient mice. Two experiments contained 2 doses of antigen and two experiments contained 1 antigen dose. Antigen doses ranged from 0.22 µM–22 µM, using 100 µl. Each point represents the mean of 2–3 mice for that condition. Mean ± s.e.m. are shown in <i>A</i> and <i>B</i>. A two-tailed pair-wise student t-test was used for statistical analyses in <i>C</i>.</p

<i>In vitro</i> cross-presentation of a calreticulin-fused soluble antigen.

<p>(A) Gel-filtration chromatogram of <i>E. coli</i>-derived OVA or the OVA-calreticulin (OVA- CRT) fusion protein (left). SDS-PAGE analysis of pooled fractions from left panel; proteins were loaded in equimolar amounts (right) and coomassie stained. (B) Indicated proteins were incubated with BMDC for 3 hours. BMDC were fixed and CFSE labeled OT-I T cells were added. IL-2 levels in supernatants were determined by ELISA (left panel; 24 hour time point). OT-I T cell proliferation was measured at 72 hours in response to 44 µM OVA or OVA-CRT. The solid grey profile indicates the condition where no antigen (no Ag) was added. Data are representative of two independent analyses. (C, D) OVA-CRT and OVA were labeled with allophycocyanin. (C) Labeling intensity was determined by fluorescence imaging of the proteins after separation by SDS-PAGE (inset). Fluorescence intensity was quantified for the indicated proteins. (D) Binding of fluorescent proteins to BMDC was assessed by flow cytometry. BMDC were incubated with labeled proteins on ice before being analyzed by flow cytometry. BMDC not incubated with proteins are depicted as a grey filled. Representative of two independent experiments performed with the same labeled proteins. Mean ± s.e.m. are shown in <i>B</i>.</p

Proinflammatory Signaling Regulates Hematopoietic Stem Cell Emergence

Hematopoietic stem cells (HSCs) underlie the production of blood and immune cells for the lifetime of an organism. In vertebrate embryos, HSCs arise from the unique transdifferentiation of hemogenic endothelium comprising the floor of the dorsal aorta during a brief developmental window. To date, this process has not been replicated in&nbsp;vitro from pluripotent precursors, partly because the full complement of required signaling inputs remains to be determined. Here, we show that TNFR2 via TNF? activates the Notch and NF-?B signaling pathways to establish HSC fate, indicating a requirement for inflammatory signaling in HSC generation. We determine that primitive neutrophils are the major source of TNF?, assigning a role for transient innate immune cells in establishing the HSC program. These results demonstrate that proinflammatory signaling, in the absence of infection, is utilized by the developing embryo to generate the lineal precursors of the adult hematopoietic system