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Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation.

Carroll-Portillo A, Cannon JL, te Riet J, Holmes A, Kawakami Y, Kawakami T, Cambi A, Lidke DS - J. Cell Biol. (2015)

Bottom Line: Direct cellular contact differentially regulates the secreted cytokine profile, indicating that MC modulation of DC populations is influenced by the nature of their interaction.Synapse formation requires integrin engagement and facilitates the transfer of internalized MC-specific antigen from MCs to DCs.The transferred material is ultimately processed and presented by DCs and can activate T cells.

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Affiliation: Department of Pathology, The University of New Mexico School of Medicine, Albuquerque, NM 87131.

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MC transfer of OVA antigen to DCs results in T cell activation. MC/9 MCs were activated and incubated with DCs to facilitate antigen transfer. After 1 h of transfer, cells were spun down and co-cultured with 100 ng/ml LPS and T cells for 16 h. Cells were harvested and analyzed using flow cytometry. T cells (IgE−/CD11c−/CD3+/CD4+) were assessed for CD25 up-regulation. (A) T cells co-cultured with MCs not primed with IgE but in the presence of DNP-OVA and DCs. (B) T cells co-cultured with DCs in the presence of media from IgE-DNP-OVA–activated MCs (no MCs present). (C) T cells co-cultured with IgE-DNP-OVA–activated MCs. (D) T cells co-cultured with DNP-BSA–activated MCs and DCs. (E) T cells co-cultured with 100 µg/ml DNP-OVA–activated MCs and DCs. (F) T cells co-cultured with 10 µg/ml DNP-OVA–activated MCs and DCs. (G) T cells activated by DCs directly presenting OVA antigen. (H) T cells alone. Shown is one representative experiment of two.
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fig7: MC transfer of OVA antigen to DCs results in T cell activation. MC/9 MCs were activated and incubated with DCs to facilitate antigen transfer. After 1 h of transfer, cells were spun down and co-cultured with 100 ng/ml LPS and T cells for 16 h. Cells were harvested and analyzed using flow cytometry. T cells (IgE−/CD11c−/CD3+/CD4+) were assessed for CD25 up-regulation. (A) T cells co-cultured with MCs not primed with IgE but in the presence of DNP-OVA and DCs. (B) T cells co-cultured with DCs in the presence of media from IgE-DNP-OVA–activated MCs (no MCs present). (C) T cells co-cultured with IgE-DNP-OVA–activated MCs. (D) T cells co-cultured with DNP-BSA–activated MCs and DCs. (E) T cells co-cultured with 100 µg/ml DNP-OVA–activated MCs and DCs. (F) T cells co-cultured with 10 µg/ml DNP-OVA–activated MCs and DCs. (G) T cells activated by DCs directly presenting OVA antigen. (H) T cells alone. Shown is one representative experiment of two.

Mentions: First, α-DNP-IgE–primed MC/9 MCs were activated by DNP-OVA cross-linker for 15 min, washed to remove unbound DNP-OVA, and added to imDCs. After 1 h of actMC–imDC preincubation, OTII T cells were added to the co-culture. For these experiments, 100 ng/ml Escherichia coli lipopolysaccharide (LPS) was added concurrently with T cells to facilitate DC maturation and T cell co-stimulation. The level of CD25 (IL2 receptor, α subunit) was quantified with flow cytometry to measure the level of T cell activation (Fig. 7; Depper et al., 1985). At 16 h after co-culture, the T cells displayed a significant up-regulation of CD25 when in the presence of OVA-DNP actMCs and DCs (Fig. 7 E, 27.9%). This up-regulation is dependent on antigen concentration, as a 10-fold decrease in concentration of OVA-DNP results in decreased CD25 up-regulation (Fig. 7 F, 7.2%). CD25 up-regulation was not seen in the absence of MC activation (Fig. 7 A, 1.9%) or when addition of only the supernatant from the actMC wash was added (Fig. 7 B, 1%). Furthermore, activation of MCs by DNP-BSA, which leads to MC polarization and material transfer (Fig. 4 and Fig. 5), did not induce CD25 up-regulation in OTII T cells (Fig. 7 D, 1.7%), confirming that up-regulation is antigen specific and not merely caused by cytokine release. There are studies that in vitro actMCs can express major histocompatibility class II (Grabbe et al., 1997; Kambayashi et al., 2009) and that direct interactions occur between MCs and T cells (Hershko and Rivera, 2010; Mekori and Hershko, 2012). However, DNP-OVA activation of MCs followed by direct co-incubation with OTII T cells (no DCs present) did not lead to detectable CD25 up-regulation (Fig. 7 C, 0.4%), consistent with the lack of costimulatory molecules on MCs needed to activate naive T cells directly (Kambayashi et al., 2009). These results demonstrate that material taken up by actMCs and subsequently transferred to DCs can then be processed by the DCs and presented to T cells, leading to productive T cell activation.


Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation.

Carroll-Portillo A, Cannon JL, te Riet J, Holmes A, Kawakami Y, Kawakami T, Cambi A, Lidke DS - J. Cell Biol. (2015)

MC transfer of OVA antigen to DCs results in T cell activation. MC/9 MCs were activated and incubated with DCs to facilitate antigen transfer. After 1 h of transfer, cells were spun down and co-cultured with 100 ng/ml LPS and T cells for 16 h. Cells were harvested and analyzed using flow cytometry. T cells (IgE−/CD11c−/CD3+/CD4+) were assessed for CD25 up-regulation. (A) T cells co-cultured with MCs not primed with IgE but in the presence of DNP-OVA and DCs. (B) T cells co-cultured with DCs in the presence of media from IgE-DNP-OVA–activated MCs (no MCs present). (C) T cells co-cultured with IgE-DNP-OVA–activated MCs. (D) T cells co-cultured with DNP-BSA–activated MCs and DCs. (E) T cells co-cultured with 100 µg/ml DNP-OVA–activated MCs and DCs. (F) T cells co-cultured with 10 µg/ml DNP-OVA–activated MCs and DCs. (G) T cells activated by DCs directly presenting OVA antigen. (H) T cells alone. Shown is one representative experiment of two.
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fig7: MC transfer of OVA antigen to DCs results in T cell activation. MC/9 MCs were activated and incubated with DCs to facilitate antigen transfer. After 1 h of transfer, cells were spun down and co-cultured with 100 ng/ml LPS and T cells for 16 h. Cells were harvested and analyzed using flow cytometry. T cells (IgE−/CD11c−/CD3+/CD4+) were assessed for CD25 up-regulation. (A) T cells co-cultured with MCs not primed with IgE but in the presence of DNP-OVA and DCs. (B) T cells co-cultured with DCs in the presence of media from IgE-DNP-OVA–activated MCs (no MCs present). (C) T cells co-cultured with IgE-DNP-OVA–activated MCs. (D) T cells co-cultured with DNP-BSA–activated MCs and DCs. (E) T cells co-cultured with 100 µg/ml DNP-OVA–activated MCs and DCs. (F) T cells co-cultured with 10 µg/ml DNP-OVA–activated MCs and DCs. (G) T cells activated by DCs directly presenting OVA antigen. (H) T cells alone. Shown is one representative experiment of two.
Mentions: First, α-DNP-IgE–primed MC/9 MCs were activated by DNP-OVA cross-linker for 15 min, washed to remove unbound DNP-OVA, and added to imDCs. After 1 h of actMC–imDC preincubation, OTII T cells were added to the co-culture. For these experiments, 100 ng/ml Escherichia coli lipopolysaccharide (LPS) was added concurrently with T cells to facilitate DC maturation and T cell co-stimulation. The level of CD25 (IL2 receptor, α subunit) was quantified with flow cytometry to measure the level of T cell activation (Fig. 7; Depper et al., 1985). At 16 h after co-culture, the T cells displayed a significant up-regulation of CD25 when in the presence of OVA-DNP actMCs and DCs (Fig. 7 E, 27.9%). This up-regulation is dependent on antigen concentration, as a 10-fold decrease in concentration of OVA-DNP results in decreased CD25 up-regulation (Fig. 7 F, 7.2%). CD25 up-regulation was not seen in the absence of MC activation (Fig. 7 A, 1.9%) or when addition of only the supernatant from the actMC wash was added (Fig. 7 B, 1%). Furthermore, activation of MCs by DNP-BSA, which leads to MC polarization and material transfer (Fig. 4 and Fig. 5), did not induce CD25 up-regulation in OTII T cells (Fig. 7 D, 1.7%), confirming that up-regulation is antigen specific and not merely caused by cytokine release. There are studies that in vitro actMCs can express major histocompatibility class II (Grabbe et al., 1997; Kambayashi et al., 2009) and that direct interactions occur between MCs and T cells (Hershko and Rivera, 2010; Mekori and Hershko, 2012). However, DNP-OVA activation of MCs followed by direct co-incubation with OTII T cells (no DCs present) did not lead to detectable CD25 up-regulation (Fig. 7 C, 0.4%), consistent with the lack of costimulatory molecules on MCs needed to activate naive T cells directly (Kambayashi et al., 2009). These results demonstrate that material taken up by actMCs and subsequently transferred to DCs can then be processed by the DCs and presented to T cells, leading to productive T cell activation.

Bottom Line: Direct cellular contact differentially regulates the secreted cytokine profile, indicating that MC modulation of DC populations is influenced by the nature of their interaction.Synapse formation requires integrin engagement and facilitates the transfer of internalized MC-specific antigen from MCs to DCs.The transferred material is ultimately processed and presented by DCs and can activate T cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, The University of New Mexico School of Medicine, Albuquerque, NM 87131.

Show MeSH
Related in: MedlinePlus