Requirement for CD1d expression by B cells to stimulate NKT cell–enhanced antibody production

Abstract

IntroductionCD1d is expressed by antigen-presenting cells (APCs) and presents glycolipids, including ␣-galactosylceramide (␣-GC) to CD1d-restricted natural killer-like T (NKT) cells. 1-3 NKT cells stimulate Th1 and Th2 responses in vivo and are thus implicated in asthma, infectious disease, cancer, and autoimmunity. [4][5][6][7][8] A newly appreciated aspect of NKT function is their potential to regulate, enhance, and sustain humoral immune responses against foreign Ags, including those expressed by parasites, bacteria, and viruses. [9][10][11][12][13][14][15][16][17][18][19] Despite clear demonstrations that NKT cells impact humoral immunity, the mechanisms are not defined. We therefore tested the hypothesis that CD1d glycolipid presentation by B cells in vivo was required for NKT-enhanced humoral immunity. After reconstitution of B cell-deficient MT mice with CD1d ϩ/ϩ or CD1d Ϫ/Ϫ B cells and subsequent immunization, we observed that NKTenhanced antibody (Ab) responses were deficient without CD1d expression by B cells. Our data introduce an important concept in humoral immunity: the direct interaction of B cells and NKT cells to facilitate enhanced Ab responses. Methods MiceC57Bl/6 mice were from the National Cancer Institute (Bethesda, MD) and MT mice from the Jackson Laboratory (Bar Harbor, ME). CD1d Ϫ/Ϫ mice have been described previously. 20 All procedures were approved by the Institutional Animal Care and Use Committee at University of Oklahoma Health Sciences Center and performed on female mice between 6 and 10 weeks of age. Antibodies and fluorochromesAnti-CD1d, -TCR␤, and isotype control mAbs were from BD Biosciences (San Jose, CA), anti-B220 and -CD19 mAbs from Biolegend (San Diego, CA), anti-CD4, -Thy1.2, and 2.4G2 mAbs from BioXpres (West Lebanon, NH), CD1d/␣-GC tetramers from the National Institute of Allergy and Infectious Diseases (NIAID) Tetramer Facility (Emory University, Atlanta, GA), and carboxyfluorescein diacetate succinimidyl ester (CFSE) from Invitrogen (Carlsbad, CA). Cells and adoptive transfersSpleens and thymi were subjected to mechanical disruption. Splenic erythrocytes were removed by hypotonic lysis. NKT and T cells were depleted by Lo-Tox complement-mediated lysis of anti-CD4/Thy1.2-labeled splenocytes (Cedarlane, Burlington, NC). Magnetic sorting was performed according to manufacturer's instructions using anti-fluorescein isothiocyanate microbeads in conjunction with a fluorescein isothiocyanateanti-CD19 mAb (Miltenyi Biotech, Auburn, CA). A BD FACScalibur was used to confirm cell purity. Where indicated, cells were labeled with CFSE (9 M in phosphate-buffered saline [PBS]/0.1% w/v bovine serum albumin) for 10 minutes at 37°C. This was followed by incubation for 5 minutes at 4°C and washing with PBS. Para-orbital injection was used to adoptively transfer 2 ϫ 10 7 cells into recipient mice. Immunization and sera collectionRecipient mice (MT) were immunized intraperitoneally 24 hours after adoptive transfer with either 20 g NP-KLH (Biosearch Technologies, Novato, CA) or 20 g NP-KL...

CD1d-restricted iNKT cells, the ‘Swiss-Army knife’ of the immune system

“…The mechanism was mediated through the combined action of CD40-CD40L interactions and cytokine secretion. CD1d expression by B cells is also absolutely required for the iNKT cellenhanced response, suggesting cognate interaction between iNKT cells and B cells [61]. Interestingly, while the effector antibody response is unaffected in iNKT-deficient mice, a significantly faster decay of circulating antibodies was observed, pointing to a potential role of iNKT cells in the homeostasis of plasma cells [60λ].…”

NK T cells provide lipid antigen-specific cognate help for B cells

“…Antibody production by protein-specific B cells is most efficient when the B cell receives cognate T cell help from T cells specific for peptides contained within the same protein internalized through the BcR (15), so in analogy to MHC II-restricted responses, class switched antilipid responses may depend on lipidspecific T cells. Vaccine studies show that murine MHC IIrestricted anti-protein IgG1 and IgA responses are improved by in vivo coadministration of ␣GalCer (16) and have shown a requirement for CD1d on B cells (17), but direct, cognate help from CD1d-restricted lipid-specific T cells for a lipid-specific B cell has not been described.…”

iNKT‐cell help to B cells: A cooperative job between innate and adaptive immune responses

“…It has been observed, however, that immunization of WT mice with the haptenated protein 4-hydroxy-3-nitrophenylacetylkeyhole limpet hemocyanin (NP-KLH) mixed with αGalCer elicits class-switched Ab responses that require both CD1d expression on B cells and the presence of Th cells [34,59]. At variance with the described examples of cognate and noncognate iNKT-cell help [38,39,54], the induction of B-cell responses by this particular Ag model does not require CD40L and CD1d expression on iNKT cells and DCs, respectively [60,61].…”

“…However, the GCs that are induced by iNKT FH -cell help are short-lived and do not produce longlived plasma cells and memory responses [54][55][56] [38,56]. In CD4 + T-cell-deficient mice, iNKT FH cells expanded more than in Th-cell-sufficient mice [55], suggesting that in physiological conditions the expansion of iNKT FH cells may be limited by the competition with T FH cells and/or by the suppression exerted by a specialized subset of Treg cells that have been found infiltrating GCs [57,58].It has been observed, however, that immunization of WT mice with the haptenated protein 4-hydroxy-3-nitrophenylacetylkeyhole limpet hemocyanin (NP-KLH) mixed with αGalCer elicits class-switched Ab responses that require both CD1d expression on B cells and the presence of Th cells [34,59]. At variance with the described examples of cognate and noncognate iNKT-cell help [38,39,54], the induction of B-cell responses by this particular Ag model does not require CD40L and CD1d expression on iNKT cells and DCs, respectively [60,61].…”