IgG antibodies mediate the clearance of target cells via the engagement of Fc gamma receptors (FcγRs) on effector cells by eliciting antibody-dependent cellular cytotoxicity and phagocytosis (ADCC ...and ADCP, respectively). Because (i) the IgG Fc domain binds to multiple FcγRs with varying affinities; (ii) even low Fc:FcγRs affinity interactions can play a significant role when antibodies are engaged in high avidity immune complexes and (iii) most effector cells express multiple FcγRs, the clearance mechanisms that can be mediated by individual FcγR are not well-understood. Human FcγRIIIa (hFcγRIIIa; CD16a), which exists as two polymorphic variants at position 158, hFcγRIIIa
and hFcγRIIIa
, is widely considered to only trigger ADCC, especially with natural killer (NK) cells as effectors. To evaluate the role of hFcγRIIIa ligation in myeloid-derived effector cells, and in particular on macrophages and monocytes which express multiple FcγRs, we engineered an aglycosylated engineered human Fc (hFc) variant, Fc3aV, which binds exclusively to hFcγRIIIa
. Antibodies formatted with the Fc3aV variant bind to the hFcγRIIIa
allotype with a somewhat lower K
than their wild type IgG1 counterparts, but not to any other hFcγR. The exceptional selectivity for hFcγRIIIa
was demonstrated by SPR using increased avidity, dimerized GST-fused versions of the ectodomains of hFcγRs and from the absence of binding of large immune complex (IC) to CHO cells expressing each of the hFcγRs, including notably, the FcγRIIIa
variant or the highly homologous FcγRIIIb. We show that even though monocyte-derived GM-CSF differentiated macrophages express hFcγRIIIa at substantially lower levels than the other two major activating receptors, namely hFcγRI or hFcγRIIa, Fc3aV-formatted Rituximab and Herceptin perform ADCP toward CD20- and Her2-expressing cancer cells, respectively, at a level comparable to that of the respective wild-type antibodies. We further show that hFcγRIIIa activation plays a significant role on ADCC by human peripheral monocytes. Our data highlight the utility of Fc3aV and other similarly engineered exquisitely selective, aglycosylated Fc variants toward other hFcγRs as tools for the detailed molecular understanding of hFcγR biology.
GATA-3 seals Notch-induced T cell commitment (46.1) García-Ojeda, Marcos E; Lemaître, Fabrice; Richard-Le Goff, Odile ...
The Journal of immunology (1950),
04/2009, Letnik:
182, Številka:
1_Supplement
Journal Article
Recenzirano
Abstract
Transcription factors orchestrate T-lineage differentiation in the thymus. One early checkpoint involves Notch-1 stimulation of thymic immigrants that promotes T-cell commitment at the ...expense of the B-lineage program. The zinc-finger transcription factor GATA-3 is required for T cell specification, although its mechanism of action is unknown. Here we identify molecular targets of GATA-3 in early thymocytes and show that conditional ablation of GATA-3 expression in committed pro-T-cells unmasks a latent B-cell potential that is undetectable in normal T-cell precursors. Thus, GATA-3 delimits the developmental options in early thymocyte precursors by sealing T-cell fate induced by Notch signals.
M.E. García-Ojeda was funded by a Pasteur Foundation Fellowship. This work was supported by grants from the Institut Pasteur, the Inserm and the Ligue Nationale contre le Cancer. M.E. Garcia-Ojeda and F. Lemaître contributed equally to this work.
Interferon-producing killer dendritic cells (IKDCs) are a recently described subset of CD11c super(lo)B220 super(+) cells that share phenotypic and functional properties of DCs and natural killer ...(NK) cells (Chan, C.W., E. Crafton, H.N. Fan, J. Flook, K. Yoshimura, M. Skarica, D. Brockstedt, T.W. Dubensky, M.F. Stins, L.L. Lanier, et al. 2006. Nat. Med. 12:207-213; Taieb, J., N. Chaput, C. Menard, L. Apetoh, E. Ullrich, M. Bonmort, M. Pequignot, N. Casares, M. Terme, C. Flament, et al. 2006. Nat. Med. 12:214-219). IKDC development appears unusual in that cytokines using the interleukin (IL)-2 receptor {szligbeta} (IL-2R{szligbeta}) chain but not those using the common gamma chain ( gamma sub(c)) are necessary for their generation. By directly comparing Rag2 super(-/-) gamma sub(c) super(-/y), Rag2 super(-/-)IL-2R{szligbeta} super(-/-), Rag2 super(-/-)IL-15 super(-/-), and Rag2 super(-/-)IL-2 super(-/-) mice, we demonstrate that IKDC development parallels NK cell development in its strict IL-15 dependence. Moreover, IKDCs uniformly express NK-specific Ncr-1 transcripts (encoding NKp46), whereas NKp46 super(+) cells are absent in Ncr1 super(gfp/+) gamma sub(c) super(-/y) mice. Distinguishing features of IKDCs (CD11c super(lo)B220 super(+)MHC-II super(+)) were carefully examined on developing NK cells in the bone marrow and on peripheral NK cells. As B220 expression was heterogeneous, defining B220 super(lo) versus B220 super(hi) NK1.1 super(+) NK cells could be considered as arbitrary, and few phenotypic differences were noted between NK1.1 super(+) NK cells bearing different levels of B220. CD11c expression did not correlate with B220 or major histocompatibility complex (MHC) class II (MHC-II) expression, and most MHC-II super(+) NK1.1 super(+) cells did not express B220 and were thus not IKDCs. Finally, CD11c, MHC-II, and B220 levels were up-regulated on NK1.1 super(+) cells upon activation in vitro or in vivo in a proliferation-dependent fashion. Our data suggest that the majority of CD11c super(lo)B220 super(+) "IKDC-like" cells represent activated NK cells.
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