Colt Egelston

Rush University Medical Center, Chicago, IL, United States

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Publications (7)74.81 Total impact

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    ABSTRACT: P18-I10 (RGPGRAFVTI), and OVA 257–264 (SIINFEKL) were synthesized by NeoMPS. Equimolar mixtures of the phosphorothioate CpG-ODNs 1555 (GCTAGACGTTAGCGT) and 1466 (TCAACGTTGA) (CpG sequences underlined) or control ODNs 1612 (GCTAGATGT-TAGCGT) and 1471 (TCAAGCTTGA) were synthesized at the Center for Biologics Evaluation and Research core facility. All were free of endotoxin and protein contamination. Lipoteichoic acids (LTA), macrophage-activating lipoprotein (MALP-2), zy-mosan (Zym), Pam 3 CSK 4 (Pam3), poly(inosinic acid)poly(cytidylic acid) [poly(IC) or PIC], lipopolysaccharide (LPS), monophosphoryl lipid A (MPL), and suppressive ODN 2088 were purchased from Invivogen. E6020 (a TLR4 ligand) was obtained from Eisai. Dose titration was performed to determine the working doses based on induction of minimal amounts of cytokines by individual ligands. Inhibitors for P38 (SB203580), ERK (FR180204), JNK (SP600125), and NF-B (SN50) were purchased from EMD Biosciences. Neutralizing antibodies for IL-12p70, IL-1, and IL-6, TNF were purchased from R&D Systems or eBioscience. Recombinant IL-12, TNF, MIP-1, and IP-10 proteins were purchased from Peprotech. Cell Isolation, Purification, and Coculture. Bone marrow cells were cultured at 7 10 5 per ml for 6 days in the presence of 15 ng/ml GM-CSF (Peprotech) in RPMI medium 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. DCs and macrophages were obtained from suspension and adherent cells, respectively. Popliteal lymph node (LN) cells were isolated after footpad immunization either with DCs or with peptide. For T cell purification, spleens were removed from naïve mice. Total T cells as well as CD4 and CD8 T cell subsets were separated by negative selection (to avoid perturbation) on an autoMACS Separator (Miltenyi Biotec) by using a mixture of antibodies against CD45R, CD49b, CD11b, Ter-119, and/or CD8 or CD4. The purity of sorted cell populations was at least 97%. After 20 h of stimulation with various TLR ligands, stimulator cells were washed to remove excess reagents. Purified total T cells or T cell subsets were used as responders, cocultured with syngeneic stimulators at a ratio of 1:2.5. In some experiments, neutralizing antibodies for IFN-and IFN-(R&D Systems) were included in the culture of DCs with TLR ligands.
    Proceedings of the National Academy of Sciences 01/2013; · 9.74 Impact Factor
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    ABSTRACT: Figure S1 The triple TLR ligands do not further enhance T cell responses by increasing T cell numbers. BM-DCs were pretreated with TLR ligands for 20 h. Excessive TLR ligands were washed off and DCs were cocultured with splenic Pan T cells freshly isolated from syngeneic mice for 24 h. T cell subsets stimulated to express CD69 were analyzed by flow. Results represent one of two independent experiments.
    Journal of Clinical Investigation 01/2013; · 12.81 Impact Factor
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    ABSTRACT: To determine whether myeloid cells (such as granulocytes) present in the synovial fluid (SF) of arthritic joints have an impact on adaptive immunity. Specifically, we investigated the effects of SF cells harvested from the joints of mice with proteoglycan-induced arthritis (PGIA), on dendritic cell (DC) maturation and antigen-specific T cell proliferation. We monitored DC maturation (MHCII and CD86 expression) by flow cytometry upon coculture of DCs with SF cells or spleen myeloid cells from mice with PGIA. The effects of these myeloid cells on T cell proliferation were studied using T cells purified from PG-specific T cell receptor (TCR)-transgenic (Tg) mice. Phenotype analysis of myeloid cells was performed by immunostaining, reverse transcription-polymerase chain reaction, Western blotting, and biochemical assays. Inflammatory SF cells significantly suppressed the maturation of DCs upon coculture. PG-TCR-Tg mouse T cells cultured with antigen-loaded DCs showed dramatic decreases in proliferation in the presence of SF cells. Spleen myeloid cells from arthritic mice did not have suppressive effects. SF cells were unable to suppress CD3/CD28-stimulated proliferation of the same T cells, suggesting a DC-dependent mechanism. SF cells exhibited all of the characteristics of myeloid-derived suppressor cells (MDSCs) and exerted suppression primarily through the production of nitric oxide and reactive oxygen species by granulocyte-like cells. SF in the joints of mice with PGIA contains a population of granulocytic MDSCs that potently suppress DC maturation and T cell proliferation. These MDSCs have the potential to limit the expansion of autoreactive T cells, thus breaking the vicious cycle of autoimmunity and inflammation.
    Arthritis & Rheumatology 04/2012; 64(10):3179-88. · 7.48 Impact Factor
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    ABSTRACT: Inflammatory joint destruction in rheumatoid arthritis (RA) may be triggered by autoantibodies, the production of which is supported by autoreactive T cells. Studies on RA and animal models of the disease suggest that T cells recruited in the joints can locally initiate or propagate arthritis. Herein, we investigated the role of joint-homing versus lymphoid organ-homing T cells in the development of proteoglycan-induced arthritis (PGIA), an autoimmune model of RA. To identify T cells migrating to the joints before and during development of autoimmune arthritis, we transferred fluorescence-labeled T cells, along with antigen-presenting cells, from BALB/c mice with PGIA to naïve syngeneic severe combined immunodeficient (SCID) mice. We then monitored the recruitment of donor T cells in the ankle joints and joint-draining lymph nodes of the recipients using in vivo two-photon microscopy and ex vivo detection methods. To limit T-cell access to the joints, we selectively depleted T cells in the blood circulation by treatment with FTY720, an inhibitor of lymphocyte egress from lymphoid organs. Reduction of T cell presence in both lymphoid organs and blood was achieved by injection of donor cells from which T cells were removed prior to transfer. T and B cells were quantitated by flow cytometry, and antigen (PG)-specific responses were assessed by cell proliferation and serum antibody assays. Despite development of adoptively transferred arthritis in the recipient SCID mice, we found very few donor T cells in their joints after cell transfer. Treatment of recipient mice with FTY720 left the T-cell pool in the lymphoid organs intact, but reduced T cells in both peripheral blood and joints. However, FTY720 treatment failed to inhibit PGIA development. In contrast, arthritis was not seen in recipient mice after transfer of T cell-depleted cells from arthritic donors, and serum autoantibodies to PG were not detected in this group of mice. Our results suggest that antigen-specific T cells, which home to lymphoid organs and provide help to B cells for systemic autoantibody production, play a greater role in the development and progression of autoimmune arthritis than the small population of T cells that migrate to the joints.
    Arthritis research & therapy 03/2010; 12(2):R44. · 4.27 Impact Factor
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    ABSTRACT: TLR ligands are promising candidates for the development of novel vaccine adjuvants that can elicit protective immunity against emerging infectious diseases. Adjuvants have been used most frequently to increase the quantity of an immune response. However, the quality of a T cell response can be more important than its quantity. Stimulating certain pairs of TLRs induces a synergistic response in terms of activating dendritic cells and eliciting/enhancing T cell responses through clonal expansion, which increases the number of responding T cells. Here, we have found that utilizing ligands for 3 TLRs (TLR2/6, TLR3, and TLR9) greatly increased the protective efficacy of vaccination with an HIV envelope peptide in mice when compared with using ligands for only any 2 of these TLRs; surprisingly, increased protection was induced without a marked increase in the number of peptide-specific T cells. Rather, the combination of these 3 TLR ligands augmented the quality of the T cell responses primarily by amplifying their functional avidity for the antigen, which was necessary for clearance of virus. The triple combination increased production of DC IL-15 along with its receptor, IL-15Ralpha, which contributed to high avidity, and decreased expression of programmed death-ligand 1 and induction of Tregs. Therefore, selective TLR ligand combinations can increase protective efficacy by increasing the quality rather than the quantity of T cell responses.
    The Journal of clinical investigation 02/2010; 120(2):607-16. · 15.39 Impact Factor
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    ABSTRACT: Figure S1 The triple TLR ligands do not further enhance T cell responses by increasing T cell numbers. BM-DCs were pretreated with TLR ligands for 20 h. Excessive TLR ligands were washed off and DCs were cocultured with splenic Pan T cells freshly isolated from syngeneic mice for 24 h. T cell subsets stimulated to express CD69 were analyzed by flow. Results represent one of two independent experiments.
    The Journal of clinical investigation 01/2010; 120(2):607-616. · 15.39 Impact Factor
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    ABSTRACT: Toll-like receptors (TLRs) may need to cooperate with each other to be effective in detecting imminent infection and trigger immune responses. Understanding is still limited about the intracellular mechanism of this cooperation. We found that when certain TLRs are involved, dendritic cells (DCs) establish unidirectional intracellular cross-talk, in which the MyD88-independent TRIF-dependent pathway amplifies the MyD88-dependent DC function through a JNK-dependent mechanism. The amplified MyD88-dependent DC function determines the induction of the T cell response to a given vaccine in vivo. Therefore, our study revealed an underlying TLR mechanism governing the functional, nonrandom interplay among TLRs for recognition of combinatorial ligands that may be dangerous to the host, providing important guidance for design of novel synergistic molecular vaccine adjuvants.
    Proceedings of the National Academy of Sciences 11/2008; 105(42):16260-5. · 9.74 Impact Factor