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1.  Correction: Targeting Leishmania major Antigens to Dendritic Cells In Vivo Induces Protective Immunity 
PLoS ONE  2013;8(9):10.1371/annotation/5149bf8e-3843-4865-a726-0ca2820ee8f8.
doi:10.1371/annotation/5149bf8e-3843-4865-a726-0ca2820ee8f8
PMCID: PMC3782404  PMID: 24086207
2.  Targeting Leishmania major Antigens to Dendritic Cells In Vivo Induces Protective Immunity 
PLoS ONE  2013;8(6):e67453.
Efficient vaccination against the parasite Leishmania major, the causative agent of human cutaneous leishmaniasis, requires development of type 1 T-helper (Th1) CD4+ T cell immunity. Because of their unique capacity to initiate and modulate immune responses, dendritic cells (DCs) are attractive targets for development of novel vaccines. In this study, for the first time, we investigated the capacity of a DC-targeted vaccine to induce protective responses against L. major. To this end, we genetically engineered the N-terminal portion of the stress-inducible 1 protein of L. major (LmSTI1a) into anti-DEC205/CD205 (DEC) monoclonal antibody (mAb) and thereby delivered the conjugated protein to DEC+ DCs in situ in the intact animal. Delivery of LmSTI1a to adjuvant-matured DCs increased the frequency of antigen-specific CD4+ T cells producing IFN-γ+, IL-2+, and TNF-α+ in two different strains of mice (C57BL/6 and Balb/c), while such responses were not observed with the same doses of a control Ig-LmSTI1a mAb without receptor affinity or with non-targeted LmSTI1a protein. Using a peptide library for LmSTI1a, we identified at least two distinct CD4+ T cell mimetopes in each MHC class II haplotype, consistent with the induction of broad immunity. When we compared T cell immune responses generated after targeting DCs with LmSTI1a or other L. major antigens, including LACK (Leishmania receptor for activated C kinase) and LeIF (Leishmania eukaryotic ribosomal elongation and initiation factor 4a), we found that LmSTI1a was superior for generation of IFN-γ-producing CD4+ T cells, which correlated with higher protection of susceptible Balb/c mice to a challenge with L. major. For the first time, this study demonstrates the potential of a DC-targeted vaccine as a novel approach for cutaneous leishmaniasis, an increasing public health concern that has no currently available effective treatment.
doi:10.1371/journal.pone.0067453
PMCID: PMC3694010  PMID: 23840706
3.  Treml4, an Ig superfamily member, mediates presentation of several antigens to T cells in vivo, including protective immunity to HER2 protein 
Members of the Trem receptor family (Triggering receptor expressed on myeloid cells) fine-tune inflammatory responses. We previously identified one of these receptors, called Trem-like 4 (Treml4), expressed mainly in the spleen, and at high levels by CD8α+ DCs and macrophages. Like other Trem family members, Treml4 has an immunoglobulin-like extracellular domain and a short cytoplasmic tail that associates with the adaptor DAP12. To follow our initial results that Treml4-Fc fusion proteins bind necrotic cells, we now generated a knock out mouse to assess the role of Treml4 in the uptake and presentation of dying cells in vivo. Loss of Treml4 expression did not impair uptake of dying cells by CD8α+ DCs or cross-presentation of cell-associated antigen to CD8+ T cells, suggesting overlapping function between Treml4 and other receptors in vivo. To further investigate Treml4 function, we took advantage of a newly generated mAb against Treml4, and engineered its heavy chain to express 3 different antigens, i.e., ovalbumin, HIV GAGp24 and the extracellular domain of the breast cancer protein HER2. Ovalbumin directed to Treml4 was efficiently presented to CD8+ and CD4+ T cells in vivo. Anti-Treml4-GAGp24 mAbs, given along with a maturation stimulus, induced Th1 antigen-specific responses which were not observed in Treml4 knock out mice. Also, HER2 targeting using anti-Treml4 mAbs elicited combined CD4+ and CD8+ T cell immunity, and both T cells participated in resistance to a transplantable tumor. Therefore, Treml4 participates in antigen presentation in vivo, and targeting antigens with anti-Treml4 antibodies enhances immunization of otherwise naïve mice.
doi:10.4049/jimmunol.1102541
PMCID: PMC3262937  PMID: 22210914
Dendritic cells; Monocytes/Macrophages; Antibodies; Antigen presentation
4.  Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209+ dendritic cells for immune T cell areas 
Cell  2010;143(3):416-429.
SUMMARY
Dendritic cells (DCs), critical antigen presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated Mo-DCs develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen presenting function, Mo-DCs are as active as classical DCs, including cross presentation of proteins and live gram negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN+ cells with critical functions of DCs.
doi:10.1016/j.cell.2010.09.039
PMCID: PMC3150728  PMID: 21029863
5.  New monoclonal anti-mouse DC-SIGN antibodies reactive with acetone-fixed cells 
Journal of immunological methods  2010;360(1-2):66-75.
Mouse DC-SIGN CD209a is a type II transmembrane protein, one of a family of C-type lectin genes syntenic and homologous to human DC-SIGN. Current anti-mouse DC-SIGN monoclonal antibodies (MAbs) are unable to react with DC-SIGN in acetone fixed cells, limiting the chance to visualize DC-SIGN in tissue sections. We first produced rabbit polyclonal PAb-DSCYT14 against a 14-aa peptide in the cytosolic domain of mouse DC-SIGN, and it specifically detected DC-SIGN and not the related lectins, SIGN-R1 and SIGN-R3 expressed in transfected CHO cells. MAbs were generated by immunizing rats and DC-SIGN knockout mice with the extracellular region of mouse DC-SIGN.. Five rat IgG2a or IgM MAbs, named BMD10, 11, 24, 25, and 30, were selected and each MAb specifically detected DC-SIGN by FACS and Western blots, although BMD25 was cross-reactive to SIGN-R1. Two mouse IgG2c MAbs MMD2 and MMD3 interestingly bound mouse DC-SIGN but at 10 fold higher levels than the rat MAbs. When the binding epitopes of the new BMD and two other commercial rat anti-DC-SIGN MAbs, 5H10 and LWC06, were examined by competition assays, the epitopes of BMD11, 24, and LWC06 were identical or closely overlapping while BMD10, 30, and 5H10 were shown to bind different epitopes. MMD2 and MMD3 epitopes were on a 3rd noncompeting region of mouse DC-SIGN. DC-SIGN expressed on the cell surface was sensitive to collagenase treatment, as monitored by polyclonal and MAb. These new reagents should be helpful to probe the biology of DC-SIGN in vivo.
doi:10.1016/j.jim.2010.06.006
PMCID: PMC2924951  PMID: 20558171
Monoclonal Antibody; Polyclonal Antibody; DC-SIGN; CD209a; Dendritic Cells
6.  Dendritic cells require a systemic type I interferon response to mature and induce CD4+ Th1 immunity with poly IC as adjuvant 
The Journal of Experimental Medicine  2009;206(7):1589-1602.
Relative to several other toll-like receptor (TLR) agonists, we found polyinosinic:polycytidylic acid (poly IC) to be the most effective adjuvant for Th1 CD4+ T cell responses to a dendritic cell (DC)–targeted HIV gag protein vaccine in mice. To identify mechanisms for adjuvant action in the intact animal and the polyclonal T cell repertoire, we found poly IC to be the most effective inducer of type I interferon (IFN), which was produced by DEC-205+ DCs, monocytes, and stromal cells. Antibody blocking or deletion of type I IFN receptor showed that IFN was essential for DC maturation and development of CD4+ immunity. The IFN-AR receptor was directly required for DCs to respond to poly IC. STAT 1 was also essential, in keeping with the type I IFN requirement, but not type II IFN or IL-12 p40. Induction of type I IFN was mda5 dependent, but DCs additionally used TLR3. In bone marrow chimeras, radioresistant and, likely, nonhematopoietic cells were the main source of IFN, but mda5 was required in both marrow–derived and radioresistant host cells for adaptive responses. Therefore, the adjuvant action of poly IC requires a widespread innate type I IFN response that directly links antigen presentation by DCs to adaptive immunity.
doi:10.1084/jem.20090247
PMCID: PMC2715098  PMID: 19564349

Results 1-6 (6)