This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. M. tuberculosis (MTB) cell wall glycolipids (phosphatidylinositol mannoside, PIM) and lipoglycans (lipoarabinomannan, LAM) are potent virulence factors and contribute to MTB survival in host macrophages, by inhibiting innate and adaptive immune responses. Abs specific for PIM and LAM confer protection against MTB. PIM and LAM are Ags that specifically bind CD1d and CD1b, to activate CD1d- or CD1b-restricted T cells. CD1d-restricted T cells (Natural Killer T cells, NKT) provide CD1d-dependent help for Ab production. This suggests that the B cell antigen receptor (BCR) may capture and internalize PIM, to facilitate CD1d-dependent NKT activation, and subsequent Ab production.
SPECIFIC AIM 1 : Does the BCR facilitate enhanced presentation of glycolipid Ag by CD1? Experimental Progress: We tested the hypothesis that BCR-targeting of a biotin-modified CD1d-binding Ag alpha-Galactosylceramide (biotin-alpha-GalCer, Ag) led to in enhanced CD1d presentation as compared to presentation of non-targeted Ag. This novel Ag was synthesized in collaboration with Dr. Besra (University of Birmingham, U.K.). Presentation of BCR-targeted Ag to NKT cells was enhanced 100-fold compared to non-targeted Ag. CD1d presentation of BCR-targeted Ag was observed after 4 hours Ag pulse, consistent with a requirement for endosomal trafficking of BCR/Ag. Fixing APCs before Ag pulse prevented presentation of BCR-targeted but not non-targeted Ag. Blocking BCR signaling with the Syk kinase inhibitor, piceatannol, inhibited presentation of BCR-targeted Ag but not non-targeted Ag. Piceatannol blocked BCR transport to CD1d-containing vesicles, showing that intersection of BCR-targeted Ag with intracellular CD1d-containing vesicles is required for Ag presentation. Our data suggest that the BCR facilitates capture of low quantities of glycolipid Ag to enhance CD1d-dependent NKT activation. In collaboration with Dr. Reinhold (Consortium PI, UNH), we have performed mass spectrometry analysis of CD1d-containing endosomes following a pulse with BCR-targeted or non-targeted Ag. Surprisingly, the Ag was not detected, suggesting that such low levels of glycolipid Ag stimulate NKT activation, as to be undetectable by mass spectrometry. We are continuing to attempt detection of these antigens intracellularly by mass spectrometry. Productivity: We have completed our experiments on BCR-mediated antigen presentation and published the findings in International Immunology (in press).
SPECIFIC AIM 2 : Does CD1 Ag presentation represent a novel pathway for Ab production? Experimental Progress: We tested the hypothesis that alpha-GalCer stimulates Ab production in vivo in the absence of class II/TCR cognate interactions. We immunized class II-/- mice s.c. with OVA, alpha-GalCer or alpha-GalCer plus OVA, and did not observe any increase in OVA-specific serum Ab titers. When anti-CD40 agonistic mAbs were injected 24 hours following Ag (OVA plus alpha-GalCer) immunization, we observed increased OVA- but not HEL-specific Ab titers, indicating that Ab responses were also Ag specific. Immunization with OVA and alpha-GalCer separately (into opposing flanks), did not lead to Ab production, indicating physical association of pre-mixed OVA and alpha-GalCer and BCR internalization of OVA/alpha-GalCer complexes. Preliminary data also shows that immunization with PIM, followed by anti-CD40 mAb results in Ab production in class II-/- mice. Our findings show that NKT cells contribute to production of Ab reactive to protein and glycolipid Ags in vivo. Productivity: These studies form the basis for a new manuscript. To complete our studies, we require a CD1d/classII-/- double knockout to determine if Ab production is defective in the absence of CD1d and class II. We have obtained CD1-/- mice (Dr. Exley, Harvard Medical School) and COBRE Core C is generating the necessary ?double knockout? controls. CURRENT/IMMINENT DIRECTIONS: Our COBRE-funded experiments show that NKT cells provide help for Ab production, and suggest that Ab production requires glycolipid-specific BCR capture of Ag and subsequent CD1d presentation to NKT cells. Our findings were used to develop an NIH R21 grant proposal (submitted Feb 2005) in collaboration with Dr. Erickson (Project 4 leader). In that application, we proposed the hypothesis that BCR capture of MTB-derived PIM by CD1d-expressing marginal zone B cells results in CD1d/NKT-dependent anti-PIM Ab production. I am currently working to obtain new data using PIM to incorporate into an NIH grant proposal (RO1 for June 2005 deadline).
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