T cells can use either T cell receptor or CD28 receptors to absorb and internalize cell surface molecules derived from antigen-presenting cells

Abstract

At the site of contact between T cells and antigen-presenting cells (APCs), T cell receptor (TCR)-peptide-major histocompatibility complex (MHC) interaction is intensified by interactions between other molecules, notably by CD28 and lymphocyte function-associated antigen 1 (LFA-1) on T cells interacting with B7 (B7-1 and B7-2), and intracellular adhesion molecule 1 (ICAM-1), respectively, on APCs. Here, we show that during T cell-APC interaction, T cells rapidly absorb various molecules from APCs onto the cell membrane and then internalize these molecules. This process is dictated by at least two receptors on T cells, namely CD28 and TCR molecules. The biological significance of T cell uptake of molecules from APCs is unclear. One possibility is that this process may allow activated T cells to move freely from one APC to another and eventually gain entry into the circulation.

Figure 1

Expression of B7-1 and B7-2 on 2C CD8⁺ cells cultured with Dros. APCs. (A) Surface staining of purified resting 2C CD8⁺ T cells. Cells were stained with PE-conjugated anti-CD28, anti–B7-1, and anti–B7-2 specific mAbs. Isotype control staining is shown as a dotted line and staining with mAbs as a solid line. (B) Surface staining of 2C CD8⁺ cells after overnight culture with the indicated Dros. APCs loaded with 1 μM QL9 peptide. After culture the cells were stained for CD8 and for the markers indicated. The data refer to staining of gated CD8⁺ cells. Dros. APCs were gated out on the basis of their CD8⁻ phenotype and their pattern of forward and side scatter. (C) Expression of B7-1 and B7-2 mRNA by 2C cells before and after culture with Dros. APCs plus QL9 peptide. After culture 2C cells were depleted of Dros. APCs by sequential Percoll and Ficoll gradient centrifugation. Relative levels of mRNA were measured by semiquantitative reverse transcription PCR analysis. (D) Surface staining of 2C T cells after overnight culture with LPS-stimulated BALB/c (H2^d) B cell blasts.

Figure 1

Expression of B7-1 and B7-2 on 2C CD8⁺ cells cultured with Dros. APCs. (A) Surface staining of purified resting 2C CD8⁺ T cells. Cells were stained with PE-conjugated anti-CD28, anti–B7-1, and anti–B7-2 specific mAbs. Isotype control staining is shown as a dotted line and staining with mAbs as a solid line. (B) Surface staining of 2C CD8⁺ cells after overnight culture with the indicated Dros. APCs loaded with 1 μM QL9 peptide. After culture the cells were stained for CD8 and for the markers indicated. The data refer to staining of gated CD8⁺ cells. Dros. APCs were gated out on the basis of their CD8⁻ phenotype and their pattern of forward and side scatter. (C) Expression of B7-1 and B7-2 mRNA by 2C cells before and after culture with Dros. APCs plus QL9 peptide. After culture 2C cells were depleted of Dros. APCs by sequential Percoll and Ficoll gradient centrifugation. Relative levels of mRNA were measured by semiquantitative reverse transcription PCR analysis. (D) Surface staining of 2C T cells after overnight culture with LPS-stimulated BALB/c (H2^d) B cell blasts.

Figure 1

Expression of B7-1 and B7-2 on 2C CD8⁺ cells cultured with Dros. APCs. (A) Surface staining of purified resting 2C CD8⁺ T cells. Cells were stained with PE-conjugated anti-CD28, anti–B7-1, and anti–B7-2 specific mAbs. Isotype control staining is shown as a dotted line and staining with mAbs as a solid line. (B) Surface staining of 2C CD8⁺ cells after overnight culture with the indicated Dros. APCs loaded with 1 μM QL9 peptide. After culture the cells were stained for CD8 and for the markers indicated. The data refer to staining of gated CD8⁺ cells. Dros. APCs were gated out on the basis of their CD8⁻ phenotype and their pattern of forward and side scatter. (C) Expression of B7-1 and B7-2 mRNA by 2C cells before and after culture with Dros. APCs plus QL9 peptide. After culture 2C cells were depleted of Dros. APCs by sequential Percoll and Ficoll gradient centrifugation. Relative levels of mRNA were measured by semiquantitative reverse transcription PCR analysis. (D) Surface staining of 2C T cells after overnight culture with LPS-stimulated BALB/c (H2^d) B cell blasts.

Figure 1

Expression of B7-1 and B7-2 on 2C CD8⁺ cells cultured with Dros. APCs. (A) Surface staining of purified resting 2C CD8⁺ T cells. Cells were stained with PE-conjugated anti-CD28, anti–B7-1, and anti–B7-2 specific mAbs. Isotype control staining is shown as a dotted line and staining with mAbs as a solid line. (B) Surface staining of 2C CD8⁺ cells after overnight culture with the indicated Dros. APCs loaded with 1 μM QL9 peptide. After culture the cells were stained for CD8 and for the markers indicated. The data refer to staining of gated CD8⁺ cells. Dros. APCs were gated out on the basis of their CD8⁻ phenotype and their pattern of forward and side scatter. (C) Expression of B7-1 and B7-2 mRNA by 2C cells before and after culture with Dros. APCs plus QL9 peptide. After culture 2C cells were depleted of Dros. APCs by sequential Percoll and Ficoll gradient centrifugation. Relative levels of mRNA were measured by semiquantitative reverse transcription PCR analysis. (D) Surface staining of 2C T cells after overnight culture with LPS-stimulated BALB/c (H2^d) B cell blasts.

Figure 2

CD28-dependent absorption of B7-1 molecules by T cells after incubation with Dros. APCs. Resting (A) and preactivated (B) purified T cells prepared from normal B6 (CD28⁺) or CD28^−/− B6 mice were surface stained for CD28 and B7-1 before and after incubation for 1 h at 37°C with L^d.B7-1 Dros. APCs. Stained cells were examined by flow cytometry. The data show staining on gated CD4⁺ and CD8⁺ cells; contaminating Dros. APCs were excluded on the basis of forward and side scatter and by CD4 or CD8 mAb staining. Dashed lines refer to staining with an irrelevant isotype-matched control mAb.

Figure 2

CD28-dependent absorption of B7-1 molecules by T cells after incubation with Dros. APCs. Resting (A) and preactivated (B) purified T cells prepared from normal B6 (CD28⁺) or CD28^−/− B6 mice were surface stained for CD28 and B7-1 before and after incubation for 1 h at 37°C with L^d.B7-1 Dros. APCs. Stained cells were examined by flow cytometry. The data show staining on gated CD4⁺ and CD8⁺ cells; contaminating Dros. APCs were excluded on the basis of forward and side scatter and by CD4 or CD8 mAb staining. Dashed lines refer to staining with an irrelevant isotype-matched control mAb.

Figure 3

CD28-dependent T cell absorption of various cell surface molecules from Dros. APCs. Preactivated CD8⁺ cells prepared from normal B6 (A, C, and D), CD28^−/− B6 (A and D), or ICAM-1^−/− B6 mice (B) were cultured with or without M17/4 anti–LFA-1 mAb (15 μg/ml) or an isotype-matched control (Cont.) mAb (anti-B220) with Dros. APCs expressing various mouse or human (h) cell surface molecules (L^d.ICAM-1 and L^d.B7-1.ICAM-1, A and B; HLA.hB7-1.hICAM-1, C; and IA^d.B7-1, for 1 h, D) and then stained for the markers indicated; staining of T cells before culture is shown as a control. (B) An anti-B220 mAb was used as a control. In other experiments, no inhibition was seen with mAbs specific for CD8, CD27, and TCR-β. The data show staining on gated CD8⁺ cells.

Figure 3

CD28-dependent T cell absorption of various cell surface molecules from Dros. APCs. Preactivated CD8⁺ cells prepared from normal B6 (A, C, and D), CD28^−/− B6 (A and D), or ICAM-1^−/− B6 mice (B) were cultured with or without M17/4 anti–LFA-1 mAb (15 μg/ml) or an isotype-matched control (Cont.) mAb (anti-B220) with Dros. APCs expressing various mouse or human (h) cell surface molecules (L^d.ICAM-1 and L^d.B7-1.ICAM-1, A and B; HLA.hB7-1.hICAM-1, C; and IA^d.B7-1, for 1 h, D) and then stained for the markers indicated; staining of T cells before culture is shown as a control. (B) An anti-B220 mAb was used as a control. In other experiments, no inhibition was seen with mAbs specific for CD8, CD27, and TCR-β. The data show staining on gated CD8⁺ cells.

Figure 3

CD28-dependent T cell absorption of various cell surface molecules from Dros. APCs. Preactivated CD8⁺ cells prepared from normal B6 (A, C, and D), CD28^−/− B6 (A and D), or ICAM-1^−/− B6 mice (B) were cultured with or without M17/4 anti–LFA-1 mAb (15 μg/ml) or an isotype-matched control (Cont.) mAb (anti-B220) with Dros. APCs expressing various mouse or human (h) cell surface molecules (L^d.ICAM-1 and L^d.B7-1.ICAM-1, A and B; HLA.hB7-1.hICAM-1, C; and IA^d.B7-1, for 1 h, D) and then stained for the markers indicated; staining of T cells before culture is shown as a control. (B) An anti-B220 mAb was used as a control. In other experiments, no inhibition was seen with mAbs specific for CD8, CD27, and TCR-β. The data show staining on gated CD8⁺ cells.

Figure 4

TCR-dependent uptake of B7-1 and L^d from Dros. APCs by CD28^−/− (A) versus CD28⁺ (B) 2C CD8⁺ cells. Resting or preactivated CD8⁺ 2C cells (B6 background) were incubated with L^d.B7-1 Dros. APCs with or without QL9 peptide (1 μM) and then stained for B7-1 and L^d expression. Some cultures were supplemented with 1B2 or F23.2 (anti-Vβ8.2) mAb (0.25% ascites fluid). Staining of T cells before culture is shown as a control. The data show staining on gated CD8⁺ cells.

Figure 4

TCR-dependent uptake of B7-1 and L^d from Dros. APCs by CD28^−/− (A) versus CD28⁺ (B) 2C CD8⁺ cells. Resting or preactivated CD8⁺ 2C cells (B6 background) were incubated with L^d.B7-1 Dros. APCs with or without QL9 peptide (1 μM) and then stained for B7-1 and L^d expression. Some cultures were supplemented with 1B2 or F23.2 (anti-Vβ8.2) mAb (0.25% ascites fluid). Staining of T cells before culture is shown as a control. The data show staining on gated CD8⁺ cells.

Figure 5

T cell absorption of surface molecules from DCs. (A) Preactivated CD28⁺ or CD28^−/− B6 CD8⁺ cells were cultured for 1 h with purified normal B6 DCs, then treated with EDTA (1 mM) to disrupt T–DC contact and stained for expression of the markers shown. (B) Resting CD28^−/− 2C CD8⁺ cells were incubated for 3 h with B6 DCs with or without SIYR peptide (1 μM) and then stained for the markers indicated. As controls, T cells were stained before culture. The data show staining on gated CD8⁺ cells.

Figure 5

T cell absorption of surface molecules from DCs. (A) Preactivated CD28⁺ or CD28^−/− B6 CD8⁺ cells were cultured for 1 h with purified normal B6 DCs, then treated with EDTA (1 mM) to disrupt T–DC contact and stained for expression of the markers shown. (B) Resting CD28^−/− 2C CD8⁺ cells were incubated for 3 h with B6 DCs with or without SIYR peptide (1 μM) and then stained for the markers indicated. As controls, T cells were stained before culture. The data show staining on gated CD8⁺ cells.

Figure 6

Transfer of cell surface molecules from mouse APCs to rat T cells. (A) Purified resting Lewis rat T cells were cultured for 20 h with purified DBA/2 (H2^d) DCs without peptide, treated with EDTA (see the legend to Fig. 5), and then stained for the markers shown. The data show staining on gated rCD8⁺ cells; staining of fresh T cells and T cells cultured with PMA and ionomycin is shown as a control. (B) Purified rat T cells were transferred to irradiated SCID mice, recovered from spleen, LN, or thoracic duct lymph 3 d later, and stained for the markers indicated. These data show staining on gated rCD4⁺ and rCD8⁺ cells.

Figure 6

Transfer of cell surface molecules from mouse APCs to rat T cells. (A) Purified resting Lewis rat T cells were cultured for 20 h with purified DBA/2 (H2^d) DCs without peptide, treated with EDTA (see the legend to Fig. 5), and then stained for the markers shown. The data show staining on gated rCD8⁺ cells; staining of fresh T cells and T cells cultured with PMA and ionomycin is shown as a control. (B) Purified rat T cells were transferred to irradiated SCID mice, recovered from spleen, LN, or thoracic duct lymph 3 d later, and stained for the markers indicated. These data show staining on gated rCD4⁺ and rCD8⁺ cells.

Figure 7

Fate of B7-1 molecules absorbed by T cells. (A) Top, Preactivated B6 CD8⁺ cells were cultured with the indicated Dros. APCs in the absence of peptide for 10 min. T–APC conjugates were detected by FACS^® analysis on the basis of combined CD8 expression and high side scatter (SSC). Percentage of CD8⁺ cells forming the conjugates with Dros. APCs was plotted. Bottom, Preactivated B6 CD8⁺ cells were cultured with L^d.B7-1 Dros. APCs for 1 h, then depleted of APCs by cell sorting for CD8 expression and cultured alone (at 37°C) for the intervals indicated before surface staining for B7-1. (B) Preactivated CD28⁺ and CD28^−/− B6 CD8⁺ cells were incubated for 1 h with L^d.B7-1 Dros. APCs (without peptide), fixed, and stained for B7-1 (green, FITC) and CD8 (blue, Cy5). Two experiments are shown: (top) after culture in normal medium, cells were disrupted and then fixed and stained after adherence to poly-l-lysine–coated coverslips; (bottom) cells were cultured initially on poly-l-lysine–coated coverslips and then fixed and stained without disruption. (C) Preactivated or resting CD28⁺ and CD28^−/− CD8⁺ 2C cells were incubated for 1 h with L^d.B7-1 Dros. APCs with or without QL9 peptide, stained for B7-1 and CD8 as in B, and then fixed. The data in the right panels show representative surface and intracellular staining seen in multiple slices of two representative cells as detected by confocal microscopy (see Materials and Methods). Nomarski images of the cells are shown on the left. In other experiments, staining the cells for B7 with two different fluorochromes showed that B7 staining before permeabilization was restricted to the cell membrane, whereas B7 staining after permeabilization was largely intracellular.

Figure 7

Fate of B7-1 molecules absorbed by T cells. (A) Top, Preactivated B6 CD8⁺ cells were cultured with the indicated Dros. APCs in the absence of peptide for 10 min. T–APC conjugates were detected by FACS^® analysis on the basis of combined CD8 expression and high side scatter (SSC). Percentage of CD8⁺ cells forming the conjugates with Dros. APCs was plotted. Bottom, Preactivated B6 CD8⁺ cells were cultured with L^d.B7-1 Dros. APCs for 1 h, then depleted of APCs by cell sorting for CD8 expression and cultured alone (at 37°C) for the intervals indicated before surface staining for B7-1. (B) Preactivated CD28⁺ and CD28^−/− B6 CD8⁺ cells were incubated for 1 h with L^d.B7-1 Dros. APCs (without peptide), fixed, and stained for B7-1 (green, FITC) and CD8 (blue, Cy5). Two experiments are shown: (top) after culture in normal medium, cells were disrupted and then fixed and stained after adherence to poly-l-lysine–coated coverslips; (bottom) cells were cultured initially on poly-l-lysine–coated coverslips and then fixed and stained without disruption. (C) Preactivated or resting CD28⁺ and CD28^−/− CD8⁺ 2C cells were incubated for 1 h with L^d.B7-1 Dros. APCs with or without QL9 peptide, stained for B7-1 and CD8 as in B, and then fixed. The data in the right panels show representative surface and intracellular staining seen in multiple slices of two representative cells as detected by confocal microscopy (see Materials and Methods). Nomarski images of the cells are shown on the left. In other experiments, staining the cells for B7 with two different fluorochromes showed that B7 staining before permeabilization was restricted to the cell membrane, whereas B7 staining after permeabilization was largely intracellular.

Figure 7

Fate of B7-1 molecules absorbed by T cells. (A) Top, Preactivated B6 CD8⁺ cells were cultured with the indicated Dros. APCs in the absence of peptide for 10 min. T–APC conjugates were detected by FACS^® analysis on the basis of combined CD8 expression and high side scatter (SSC). Percentage of CD8⁺ cells forming the conjugates with Dros. APCs was plotted. Bottom, Preactivated B6 CD8⁺ cells were cultured with L^d.B7-1 Dros. APCs for 1 h, then depleted of APCs by cell sorting for CD8 expression and cultured alone (at 37°C) for the intervals indicated before surface staining for B7-1. (B) Preactivated CD28⁺ and CD28^−/− B6 CD8⁺ cells were incubated for 1 h with L^d.B7-1 Dros. APCs (without peptide), fixed, and stained for B7-1 (green, FITC) and CD8 (blue, Cy5). Two experiments are shown: (top) after culture in normal medium, cells were disrupted and then fixed and stained after adherence to poly-l-lysine–coated coverslips; (bottom) cells were cultured initially on poly-l-lysine–coated coverslips and then fixed and stained without disruption. (C) Preactivated or resting CD28⁺ and CD28^−/− CD8⁺ 2C cells were incubated for 1 h with L^d.B7-1 Dros. APCs with or without QL9 peptide, stained for B7-1 and CD8 as in B, and then fixed. The data in the right panels show representative surface and intracellular staining seen in multiple slices of two representative cells as detected by confocal microscopy (see Materials and Methods). Nomarski images of the cells are shown on the left. In other experiments, staining the cells for B7 with two different fluorochromes showed that B7 staining before permeabilization was restricted to the cell membrane, whereas B7 staining after permeabilization was largely intracellular.