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TCR-Mediated Internalization of Peptide-MHC Complexes Acquired by T Cells

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Science  29 Oct 1999:
Vol. 286, Issue 5441, pp. 952-954
DOI: 10.1126/science.286.5441.952

Abstract

Peptide–major histocompatibility complex protein complexes (pMHCs) on antigen-presenting cells (APCs) are central to T cell activation. Within minutes of peptide-specific T cells interacting with APCs, pMHCs on APCs formed clusters at the site of T cell contact. Thereafter, these clusters were acquired by T cells and internalized through T cell receptor–mediated endocytosis. During this process, T cells became sensitive to peptide-specific lysis by neighboring T cells (fratricide). This form of immunoregulation could explain the “exhaustion” of T cell responses that is induced by high viral loads and may serve to down-regulate immune responses.

T cell responses are initiated by T cell receptor (TCR) recognition of pMHCs on APCs (1). Upon specific interaction of T cells with APCs, TCR and MHC molecules are assembled at the center of supramolecular activation clusters (2). The fate of these TCR-MHC clusters at the T cell–APC contact site is unclear. However, it is known that interaction of TCRs with pMHC complexes is followed by TCR down-regulation (3) and that T cell–APC interaction can cause APC-derived surface molecules to adhere to the surface of T cells (4).

To investigate the fate of MHC clusters at the T cell–APC contact sites, we used Drosophila, RMA-S, and dendritic cell lines expressing MHC class I (Ld)–green fluorescent protein fusion molecules (Ld-GFP) (5) as APCs to activate CD8 T cells from the 2C TCR transgenic mouse line (6, 7). 2C T cells recognize Ld plus either QL9 peptide or a closely related peptide, p2Ca (8). Empty Ld-GFP molecules expressed on the surface of RMA-S and Drosophila cells can be efficiently loaded with exogenous peptides (7,9). Within 5 min of 2C T cells interacting with eitherDrosophila cells (Fly.Ld-GFP) or RMA-S cells (RMA-S.Ld-GFP) plus the QL9 peptide, Ld-GFP molecules formed large clusters at the T cell–APC contact site [Fig. 1A and Web figure 1 (10)]. After 30 min, the Ld-GFP clusters at the T cell–APC interface decreased in size, and small aggregates of Ld-GFP appeared concomitantly within 2C T cells at sites distal to the contact site [Fig. 1, B and C, Web figure 2 (10)]. This process was not seen with P1A (11) (Fig. 1D), a control peptide that binds to Ld but is not recognized by 2C TCR (7). Aggregates of Ld-GFP were also detected inside 2C T cells after interaction with the Ld-GFP–transfected dendritic cell line DC2.4 (12) (Fig. 1E), which presents endogenous p2Ca peptide. To exclude the possibility that GFP itself could mediate the acquisition of Ld by T cells, we used Drosophila cells expressing untagged Ld (no GFP) loaded with QL9 peptide as APCs. After culture for 1 hour, multiple aggregates of Ldmolecules were detected in 2C T cells by intracellular staining with monoclonal antibody (mAb) to Ld (13) (Fig. 1F). Thus, in the presence of antigenic peptides, Ld molecules can be acquired from APCs by T cells, and this acquisition is independent of GFP.

Figure 1

Acquisition of APC-derived MHC class I molecules by T cells. (A to E) CD8 2C T cells were cultured with APCs expressing Ld-GFP. GFP fluorescence and differential interference contrast (DIC) images obtained with a confocal microscope system (FluoView, Olympus) were overlaid. (A and B) GFP fluorescence and DIC images of a pair of resting 2C T cell-Fly.Ld-GFP loaded with QL9 peptide were acquired every 30 s for 45 min. Images at 5 and 30 min are shown. (C) Image of one pair of activated 2C T cell-RMA-S.Ld-GFP with QL9 peptide after 30 min of culture. (D) One pair of resting 2C T cell-Fly.Ld-GFP with P1A peptide. (E) Image of an activated 2C T cell after culture for 45 min with the Ld-GFP–transfected DC line. (F) Immunofluorescence image of one pair of activated 2C T cell-Fly.Ld. Activated 2C T cells were cultured for 1 hour with Drosophila cells expressing untagged Ld in the presence of QL9 peptide. Cells were fixed, permeabilized, and stained with biotinylated mAb to Ld, followed with strepavidin–Texas Red. (G) Peptide-specific immunoprecipitation of APC-derived Ld from resting 2C T cells. 2C T cells (lanes 1, 2, and 3: no T cells, 2 × 107 T cells, and 4 × 107 T cells, respectively) were cultured for 4 hours with 3 × 106 35S-methionine–labeled L-Ld cells. Lysates were prepared and immunoprecipitated with mAbs to H2K or to Ld. (H) Immunoprecipitation of Ldmolecules from 2C T cells cultured with L-Ld cells plus QL9 peptide for 4 hours with or without mAbs to 2C TCR (αTCR), to Ld (αLd), or to H-2K(αH2K).

Acquisition of APC-derived Ld molecules by 2C T cells was further demonstrated by immunoprecipitation (Fig. 1, G and H). After culture with 35S-methionine–labeled fibroblasts (L cells) transfected with Ld (L-Ld) (14), 2C T cells were highly purified and immunoprecipitation was performed with mAbs to class I molecules. Immunoprecipitation of Ld from 2C T cells cultured with L-Ld plus QL9 peptide was prominent (Fig. 1G). In the presence of the control P1A peptide (11), precipitation of Ld was limited, but clearly detectable, presumably due to the presentation of endogenous p2Ca peptide in L-Ld cells. In contrast, other MHC class I molecules (Kk and Dk) expressed by L cells were not detectable in 2C T cells by immunoprecipitation (Fig. 1G). The peptide-dependent acquisition of Ld molecules by T cells could be blocked by adding mAbs either to TCR or Ld during the culture (Fig. 1H). Thus, acquisition of Ld by T cells requires a specific interaction between TCRs and pMHC complexes.

The kinetics of acquisition of APC-derived pMHC molecules by T cells was investigated by fluorescent-activated cell sorting (FACS) analysis. After culture with Fly.Ld-GFP plus peptides for 30 min, most 2C T cells were positive for Ld-GFP with QL9 peptide but negative with the control P1A peptide (Fig. 2A). The amount of Ld-GFP on 2C T cells remained high for 2 hours, then gradually declined over 6 hours (Fig. 2B). Peptide-titration studies showed that the amount of Ld-GFP acquired by 2C T cells correlated with the concentration of QL9 peptide used in the culture (Fig. 2C). With p2Ca, which is a lower affinity peptide than QL9, acquisition of Ld-GFP was substantial but less marked than with QL9. Similar results were obtained by FACS analysis of 2C T cells stained with mAb to Ld (13) after culture withDrosophila cells expressing untagged Ld(Fly.Ld) (Fig. 2, B and C). Thus, acquisition of Ld molecules by 2C T cells is dependent on time and specific peptide concentration.

Figure 2

Time- and peptide dose–dependent acquisition of APC-derived MHC molecules and TCR down-regulation. Resting 2C T cells were cultured for the indicated time with Fly.Ld -GFP APCs plus the indicated doses of peptides. The amount of Ld-GFP acquired by CD8 2C T cells was directly analyzed by FACS. For the surface level of Ld and TCRs, 2C T cells were cultured with Fly.Ld APCs plus the indicated doses of peptides and analyzed with mAbs to Ld (αLd) or to 2C TCR (αTCR) by FACS. (A) Histograms showing the amount of Ld-GFP and TCR on 2C T cells after culture for 30 min with Drosophila APCs plus 10 μM of QL9 or P1A peptide. (B) Kinetics of TCR down-regulation and acquisition of Ld by 2C T cells with 10 μM of the indicated peptides. (C) Peptide-dose dependent TCR down-regulation and acquisition of Ld by T cells.

Rapid acquisition of Ld molecules by T cells correlated with down-regulation of 2C TCRs (Fig. 2). Because TCR down-regulation is a result of TCR internalization by T cells (15), 2C TCRs might be cointernalized with acquired Ld. In support of this possibility, Ld-GFP colocalized with TCRs in 2C T cells that were cultured with Fly.Ld-GFP plus QL9 peptide but not P1A peptide (Fig. 3A). Ld-GFP internalized by 2C T cells partly colocalized with transferrin-containing vesicles (Fig. 3B). To follow the intracellular fate of Ld-GFP, we used LysoTracker, a red fluorescent dye that specifically accumulates in low-pH compartments of cells, such as endosomes and lysosomes (16). Ld-GFP appeared in acidic compartments of 2C T cells cultured with APCs for 3 hours (Fig. 3C). Thus, MHC molecules acquired by T cells were cointernalized with TCRs through endocytosis and localized in endosomes and lysosomes.

Figure 3

TCR-mediated endocytosis of APC- derived MHC class I molecules. (A) Intracellular colocalization of TCR and Ld-GFP in 2C T cells. After culture for 1 hour with Fly.Ld-GFP APCs plus QL9 or P1A peptide, 2C T cells were permeabilized and stained first with a mixture of biotinylated mAbs to TCR (anti-CD3ɛ, anti-TCRβ, and a clonotypic mAb, 1B2) (αTCR) and then with streptavidin–Texas Red. (B) Activated 2C T cells were cultured first with Texas Red–conjugated transferrin (5 μg/ml) and then with Fly.Ld-GFP plus the QL9 peptide for 30 min. (C) Activated CD8 2C T cells were cultured with RMA-S.Ld-GFP plus QL9 for 3 hours and then stained with 5 nM LysoTracker Red DND-99 for 10 min.

The presence of APC-derived pMHC complexes on the T cell surface, detected by FACS analysis (Fig. 2), could make these T cells susceptible to lysis by neighboring T cells with peptide-specific cytotoxic T lymphocyte (CTL) activity (fratricide). To test this possibility, we first sensitized 51Cr-labeled activated T cells with APCs expressing pMHC and then added fresh CTLs to the culture to perform a standard 51Cr-release assay. Pre-culturing 51Cr-labeled 2C T cells (H2b, Ld) with Ld-expressing APCs loaded with QL9 peptide (but not P1A peptide) rendered the labeled 2C T cells susceptible to lysis by unlabeled 2C CTLs (Fig. 4, A and B). The lysis of51Cr-labeled 2C T cells was blocked by mAbs to 2C TCR (1B2) (Fig. 4A) or to Ld (Fig. 4B). To rule out the possibility that peptides released from the killed APCs could sensitize the 2C T cells for subsequent killing, we used RMA-S cells (KbDb) loaded with a mixture of SIY peptide (8) and GP33 peptide (17) to sensitize either 2C T cells or LCMV T cells (17); 2C T cells recognize Kb-SIY but not Db-GP33, whereas LCMV T cells recognize Db-GP33 but not Kb-SIY (17). The 51Cr-labeled 2C T cells that were sensitized with RMA-S cells loaded with both peptides were lysed only by 2C CTLs and not by LCMV CTLs (Fig. 4C). Conversely,51Cr-labeled LCMV T cells sensitized with RMA-S cells plus both peptides were killed only by LCMV CTLs and not by 2C CTLs (Fig. 4D). Thus, T cells were sensitized for fratricide by pMHC complexes specifically acquired from target cells. With 51Cr-labeled 2C T cells, sensitizing the T cells for lysis required a high concentration of QL9 peptide on Ld-expressing APCs (Fig. 4). Likewise, with dendritic cells (H2d) as APCs, sensitizing 2C T cells required that the DC (which expresses endogenous p2Ca peptide) be supplemented with a high concentration of QL9 peptide.

Figure 4

Sensitization of CD8 T cells to peptide-specific lysis by CTLs. Activated T cells were labeled with51Cr and then sensitized with APCs for 1 hour. During the killing phase, fresh unlabeled CTLs were added to the culture. After 4 to 6 hours, 51Cr specifically released from the sensitized T cells was measured. (A) 51Cr-labeled 2C T cells were sensitized by Fly.Ld with QL9 or P1A peptide. During the killing phase with 2C CTL, anti-2C TCR mAb (αTCR) was added to the culture. (B) Labeled 2C T cells were sensitized by RMA-S.Ld plus QL9 peptide in the presence of anti- Ld mAb (αLd), followed by culture with 2C CTLs. (C and D) Labeled 2C T cells were sensitized with RMA-S cells loaded with a mixture of SIY and GP33 peptides, and then either 2C CTLs or LCMV CTLs were used during the killing phase. (E) Labeled 2C T cells were sensitized with RMA-S.Ld cells loaded with the indicated doses of QL9 peptide, followed by culture with 2C CTLs. (F) Labeled 2C T cells were sensitized by purified DCs (H2d) from mouse spleen with or without added QL9 peptide (10 μM), followed by culture with 2C CTLs.

Fratricide of CD8 T cells after antigen-specific T cell–APC interaction could be relevant to the finding that in vivo exposure to high doses of virus causes antigen-specific CD8 T cells to proliferate briefly and then disappear (17). The mechanism of such CTL “exhaustion” is not clear. It has been suggested that the responding T cells themselves are infected with the viruses, thus becoming targets for CTLs (18). Nevertheless, detection of viruses in CTLs is not an invariable finding (17). Our data (Fig. 4) suggest that fratricide of T cells does not require direct viral infection of T cells but can be accomplished through transfer of preformed pMHC complexes from the APC to specific T cells. On the basis of the peptide-dose experiment (Fig. 4E), T cell fratricide would only occur if a high density of pMHC was transferred to the surface of the target T cells, which could happen in cells infected by high doses of virus. Because unrestricted T cell responses upon exposure to high doses of viruses would lead to dangerous immunopathology (19), reducing the intensity of the immune responses by fratricide of T cells could be a beneficial form of immunoregulation in vivo.

  • * To whom correspondence should be addressed. E-mail: zcai{at}prius.jnj.com

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