Presentation of ovalbumin internalized via the immunoglobulin-A Fc receptor is enhanced through Fc receptor γ-chain signaling by Li Shen, Marjolein van Egmond, Karyn Siemasko, Hong Gao, Terri Wade, Mark L. Lang, Marcus Clark, Jan G. J. van de Winkel, and William F. Wade Blood Volume 97(1):205-213 January 1, 2001 ©2001 by American Society of Hematology

Presentation of IgA-OVA by FcαR/γ-chain transfectants is diminished in cells with altered γ chain.(A) IIA1.6 B cells were cotransfected with FcαR and either WT γ chain (indicated with black bars), γ chain with IIA ITAM (indicated with cross-hatched bars), o... Presentation of IgA-OVA by FcαR/γ-chain transfectants is diminished in cells with altered γ chain.(A) IIA1.6 B cells were cotransfected with FcαR and either WT γ chain (indicated with black bars), γ chain with IIA ITAM (indicated with cross-hatched bars), or γ chain with Y→ F mutation (indicated with gray bars). Transfectants and DO-11-10 OVA-specific T cells were incubated with various concentrations of NIP-haptenated OVA (NIP-OVA) opsonized with an IgA anti-NIP antibody. IL-2 secretion by the T cells was quantified as a measure of antigen presentation, as described in “Materials and methods.” (B) Nontargeted OVA is presented equally by WT or altered γ-chain transfectants. FcαR transfectants with WT γ chain (indicated with black bars), IIA ITAM γ chain (indicated with cross-hatched bars), or Y→ F γ chain (indicated with gray bars) were incubated together with DO-11-10 T cells and OVA at the concentrations indicated, and IL-2 secretion was measured as given in panel A. Similar results were obtained in 3 separate experiments. Li Shen et al. Blood 2001;97:205-213 ©2001 by American Society of Hematology

Transcription of γ chain in transfected cells Transcription of γ chain in transfected cells.Total cellular RNA was isolated from cells cotransfected with FcαR and WT or altered γ-chain cDNA. Transcription of γ chain in transfected cells.Total cellular RNA was isolated from cells cotransfected with FcαR and WT or altered γ-chain cDNA. The presence of γ-chain message was detected by RT-PCR using γ-chain–specific primers, as described in “Materials and methods.” The γ-chain–specific PCR product was detected as shown in lane 1 (γ-chain cDNA+ control), lane 4 (FcαR + WT γ chain), lane 5 (FcαR + IIA ITAM γ chain), and lane 6 (FcαR + Y → F γ chain). The γ-chain–specific product was not detected when RNA was not added (lane 2) or with parent IIA1.6 cells (lane 3). Li Shen et al. Blood 2001;97:205-213 ©2001 by American Society of Hematology

Endocytosis of multimeric IgA by FcαR/γ-chain transfectants is not diminished in cells with altered γ chain.IIA1.6 cells expressing FcαR and either WT γ chain (indicated by black bars), γ chain with IIA ITAM (indicated by cross-hatched bars), or γ chain wit... Endocytosis of multimeric IgA by FcαR/γ-chain transfectants is not diminished in cells with altered γ chain.IIA1.6 cells expressing FcαR and either WT γ chain (indicated by black bars), γ chain with IIA ITAM (indicated by cross-hatched bars), or γ chain with Y → F mutation (indicated by gray bars) were incubated with a polymeric human myeloma IgA at 4°C. After 1.5 hours, aliquots were removed, washed, and incubated at 37°C for the time period indicated. All cells were then washed and stained to detect surface-bound IgA. The results are expressed as the percentage of MFI of samples held at 4°C for 2 hours. The results are shown as the mean and SD of triplicate experiments. Li Shen et al. Blood 2001;97:205-213 ©2001 by American Society of Hematology

Catabolism of 125I-labeled anti-mIgM μ chain is diminished in transfectants with altered γ chain, but it is increased by BCR ligation.(A) FcαR on IIA1.6 cells expressing FcαR and either WT γ chain (wt), γ chain with IIA ITAM (IIA ITAM), or γ chain with Y → ... Catabolism of 125I-labeled anti-mIgM μ chain is diminished in transfectants with altered γ chain, but it is increased by BCR ligation.(A) FcαR on IIA1.6 cells expressing FcαR and either WT γ chain (wt), γ chain with IIA ITAM (IIA ITAM), or γ chain with Y → F mutation (Y → F) were ligated at 4°C with the anti-FcαR mAb My43 followed by 125I-labeled antimouse IgM μ chain. The cells were then incubated at 37°C without further treatment or with additional cross-linking of BCR with antimouse IgG. After an 8-hour incubation at 37°C, supernates were harvested, and TCA-soluble supernate counts were measured as described in “Materials and methods.” The solid bars represent the amount of TCA-soluble counts released in the absence of BCR cross-linking. The hatched bars represent the amount of TCA-soluble counts released when BCR was cross-linked. Error bars indicate range of duplicate samples. The results are representative of 3 experiments. (B) IgA catabolism is defective in altered γ-chain transfectants but is restored by BCR ligation. FcαR transfectants with WT γ chain (WT, lane 1), γ chain with IIA ITAM (IIA, lane 2), or γ chain with Y → F mutation (Y → F, lane 3) were incubated with 50 μg/mL human myeloma IgA. After 4 hours the cells were washed and lysed, and intracellular IgA and IgA catabolism products were detected by SDS-PAGE and anti-IgA Western blot. Addition of 10 μg/mL anti-MIgG to the incubation mixture did not affect IgA catabolism by the WT γ-chain transfectant (lane 2), but the addition altered IgA catabolism by the IIA ITAM (lane 4) and Y → F (lane 6) γ-chain transfectants. Three separate experiments yielded the same result. Li Shen et al. Blood 2001;97:205-213 ©2001 by American Society of Hematology

Colocalization of FcαR with lamp-1 in clustered vesicles occurs in transfectants with WT γ chain but not in those with altered γ chain.Transfectants expressing FcαR and either WT γ chain (WT gamma), γ chain with IIA ITAM (IIA ITAM), or γ chain with Y → F mu... Colocalization of FcαR with lamp-1 in clustered vesicles occurs in transfectants with WT γ chain but not in those with altered γ chain.Transfectants expressing FcαR and either WT γ chain (WT gamma), γ chain with IIA ITAM (IIA ITAM), or γ chain with Y → F mutation (Y → F) were treated with anti-FcαR IgG1 mAb and FITC anti-mIgG1 (green) at 37°C, after which they were fixed, permeabilized, and counterstained with rat anti–lamp-1 and Cy3 antirat IgG (red). Yellow staining denotes areas of colocalized FcαR and lamp-1. Li Shen et al. Blood 2001;97:205-213 ©2001 by American Society of Hematology

Tyrosine phosphorylation after FcαR cross-linking is reduced in transfectants with altered γ chain.(A) Transfectants expressing FcαR and either WT γ chain (WT), γ chain with IIA ITAM (IIA), or γ chain with Y → F mutation (Y → F) were treated as follows: (1)... Tyrosine phosphorylation after FcαR cross-linking is reduced in transfectants with altered γ chain.(A) Transfectants expressing FcαR and either WT γ chain (WT), γ chain with IIA ITAM (IIA), or γ chain with Y → F mutation (Y → F) were treated as follows: (1) medium, (2) anti-mIgM, and (3) anti-FcαR IgM mAb + anti-IgM. Tyrosine phosphorylation in whole-cell lysate was detected by SDS-PAGE and Western blot analysis with antiphosphotyrosine. Three separate experiments yielded similar results. (B) Prior to immunoblotting with antiphosphotyrosine, the same nitrocellulose membrane shown in panel A was stained with ponceau red to ascertain equivalent loading. Details of the above are described in “Materials and methods.” Li Shen et al. Blood 2001;97:205-213 ©2001 by American Society of Hematology

Defective presentation of IgA-OVA is restored by BCR ligation. IIA1 Defective presentation of IgA-OVA is restored by BCR ligation.IIA1.6 cells transfected with (A) FcαR + IIA ITAM γ chain or (B) FcαR + Y→F mutant γ chain were incubated with DO-11-10 T cells and increasing concentrations of IgA-complexed NIP-OVA (NIP-OVA) wi... Defective presentation of IgA-OVA is restored by BCR ligation.IIA1.6 cells transfected with (A) FcαR + IIA ITAM γ chain or (B) FcαR + Y→F mutant γ chain were incubated with DO-11-10 T cells and increasing concentrations of IgA-complexed NIP-OVA (NIP-OVA) with (indicated by cross-hatched bars) or without (indicated by black bars) ligation of BCR with 10 μg/mL anti-mIgG. IL-2 secretion by the T cells, a measure of antigen presentation, was assayed as described in “Materials and methods.” Similar results were obtained in 3 separate experiments. Li Shen et al. Blood 2001;97:205-213 ©2001 by American Society of Hematology

Presentation of nontargeted OVA is enhanced by BCR and FcαR/WT γ-chain cross-linking but not by cross-linking of FcαR with altered γ chain.IIA1.6 cells expressing FcαR and either (A) WT γ chain, (B) γ chain with IIA ITAM, or (C) γ chain with Y → F mutation ... Presentation of nontargeted OVA is enhanced by BCR and FcαR/WT γ-chain cross-linking but not by cross-linking of FcαR with altered γ chain.IIA1.6 cells expressing FcαR and either (A) WT γ chain, (B) γ chain with IIA ITAM, or (C) γ chain with Y → F mutation were incubated with increasing concentrations of OVA with (indicated by cross-hatched bars) or without (indicated by black bars) 10 μg/mL antimouse IgG in the presence of DO-11-10 T cells. Transfectants with FcαR and either (D) WT γ chain, (E) γ chain with IIA ITAM, or (F) γ chain with Y → F mutation were also incubated with OVA with (indicated by cross-hatched bars) or without (indicated by black bars) 1:5 diluted My43 anti-FcαR IgM hybridoma supernatant and 10 μg/mL anti– mIgM in the presence of DO-11-10 T cells. Supernatants were harvested and assayed for IL-2, a measure of antigen presentation, as described in “Materials and methods.” Numbers above histogram bars denote IL-2 titers. A difference in titer of 4-fold or more is significant. Similar results were obtained in 3 separate experiments. Li Shen et al. Blood 2001;97:205-213 ©2001 by American Society of Hematology