CD8⁺ Myelin Peptide-Specific T Cells Can Chemoattract CD4⁺ Myelin Peptide-Specific T Cells: Importance of IFN-Inducible Protein 10

Synthetic peptides

Peptides were synthesized and purified by HPLC, as previously described (16). Peptides MBP87–106 (VVHFFKNIVTPRTPPPSQGK) (17), PLP40–60 (TGTEKLIETYFSKNYQDYEYL) (18), PLP45–53 (KLIETYFSK) (7), PLP80–88 (FLYGALLLA) (6), and human cytomegalovirus gB 613–628 (PSLKIFIAGNSAYEYV) (19) were dissolved in stock solutions of PBS/50% DMSO at 1 mg/ml.

Generation and characterization of T cell lines

The generation of CD8⁺CTL lines specific for PLP80–88 plus HLA-A2 and PLP45–53 plus HLA-A3 by our laboratory was previously described using PBL from two MS patients (6, 7). These CTL lines are designated PLP80–88.1–3 and PLP45–53.1–4. The CD4⁺ T cell line GD22 specific for MBP87–106 was generated from the PBL of an MS patient, as previously described (17). Two CD4⁺ T cell lines specific for PLP40–60, designated PLP40–60.1 and PLP40.60.2, were derived from the PBL of an MS patient, as described (18).

Generation and quantitation of chemokines

CD8⁺CTL lines were used for chemokine generation by thawing frozen stocks and stimulating them for 5 to 7 days with peptide-pulsed PBL (6, 7). CTL lines were cultured in 96-well round-bottom plates at 5 × 10⁴ cells/well. APCs were HLA-A2- or HLA-A3-transfected Hmy2.C1R cells (20) that were treated with mitomycin C (200 μg/ml; Sigma Chemical Co., St. Louis, MO) and pulsed (1 μM) with the appropriate peptides for 2 h at 37°C. APCs were then washed three times with PBS and added to wells at 10⁵ cells/well. Culture medium was Iscove’s modified Dulbecco’s medium (Life Technologies, Gaithersburg, MD) with 5% heat-inactivated FCS. Supernatants were collected 48 h later and stored at −70°C until assayed. Supernatants obtained from mitomycin C-treated Hmy2.C1R cells cultured alone had no detectable chemokines, as detected in sandwich ELISA assays or in chemotaxis assays (see below).

Quantitation of chemokines was performed by sandwich ELISA. Each CTL line was tested in at least two independent experiments. MIP-1α and MIP-1β assays were performed as described by the manufacturer (R&D Systems, Minneapolis, MN). IL-16 ELISA assays were performed as previously described (13). IP-10 ELISA assays were performed with biotinylated and nonlabeled polyclonal goat anti-human IP-10 (R&D Systems) similar to previously described techniques (15). The levels of sensitivity of the ELISA assays were as follows: IP-10 = 50 pg/ml; IL-16 = 1 pg/ml; MIP-1α and MIP-1β = 15 pg/ml.

The capacity of supernatants from CD8⁺ T cells to chemoattract CD4⁺ T cells was quantitated by a chemotaxis assay using Transwell plates (Costar, Cambridge, MA). Transwell plates (Costar 3421) contained polycarbonate inserts with 5-μm pores. Transwells with inserts were loaded with 1 ml of Iscove’s modified Dulbecco’s medium with 0.25% human serum albumin (chemotaxis assay medium) for 1 h at 37°C, 5% CO₂. Wells were then aspirated and loaded with 0.8 ml of supernatants or control medium and incubated for 20 min at 37°C, 5% CO₂. CD4⁺ T cells (5 × 10⁵ in chemotaxis assay medium) in 100 μl were added to the insert well, and the Transwell plates were incubated at 37°C, 5% CO₂ for 1 h. Cells present in the lower chamber were then collected and counted using a FACScan (Becton Dickinson, San Jose, CA) for 1 min with a constant flow rate of 67 μl/min. Each sample was counted three times, and the results are expressed as the average and SD of the three counts. Results reported as percentage of cell migration were calculated as the mean percentage of cell migration of indicator cells relative to the migration in the presence of chemotaxis assay medium alone (normalized to 100% in all experiments). Statistical comparisons of the significance of cell migration data were performed by Student’s t test, assuming unequal variances.

In experiments in which chemokine-specific neutralizing antisera were used to inhibit the chemotactic activity present in supernatants, 0.8 ml of supernatants plus antisera were mixed and added to the Transwell plates and incubated for 30 min at 37°C, 5% CO₂. Indicator cells were then added to the inserts, and the Transwell plates were incubated for 1 h at 37°C, 5% CO₂. The chemokine-specific neutralizing antisera were as follows: anti-IP-10 = goat anti-human rIP-10 (15), neutralization capacity = 5 μg/ml, neutralizes 1,000 pg/ml IP-10, used at 50 μg/ml; anti-IL-16 = rabbit anti-human rIL-16 (13), neutralization capacity = 5 μg/ml, neutralizes 8,000 pg/ml IL-16, used at 5 μg/ml; anti-MIP-1α = goat anti-human rMIP-1α (R&D Systems), neutralization capacity = 5 μg/ml, neutralizes 8,000 pg/ml, used at 5 μg/ml; anti-MIP-1β = anti-human rMIP-1β (R&D Systems), neutralization capacity = 5 μg/ml, neutralizes 20,000 pg/ml, used at 5 μg/ml; and anti-mouse IgG = goat anti-mouse IgG (21), used at 50 μg/ml.

Human rIP-10 was purchased from PeproTech (Rocky Hill, NJ). Chemokine receptor 5-specific mAb 5C7 (22) was obtained through AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health from LeukoSite (Cambridge, MA).

Specificity of myelin protein-specific CD4⁺ T cell lines

The specificity of the CD4⁺ T cell line GD22 for MBP87–106 presented by HLA-DR2 has been previously reported (17). Presentation of PLP40–60 by HLA-DR4 to CD4⁺ T cell lines has also been reported (18). We have generated two such PLP40–60 CD4⁺ T cell lines (designated PLP40–60.1 and PLP40–60.2) and have determined that each can secrete IFN-γ when stimulated with APC pulsed with PLP40–60, but not with an irrelevant peptide (human cytomegalovirus gB 613–628) or no peptide (data not shown). Neither T cell line could secrete IL-4 or IL-10 when stimulated with PLP40–60 (data not shown), indicating that these T cell lines express the Th1 phenotype.

Chemokine production by CD8⁺PLP-specific CTL lines

The characterization of CD8⁺CTL lines that are specific for PLP80–88, presented by HLA-A2, and PLP45–53, presented by HLA-A3, has been previously reported (6, 7). Three PLP80–88-specific CTL lines (designated PLP80–88.1–3) and four PLP45–53-specific CTL lines (designated PLP45–53.1–4) were selected and expanded for these studies. Supernatants from each CTL line were generated by stimulation with peptide-pulsed APC for 48 h and assayed for the presence of IP-10, IL-16, MIP-1α, and MIP-1β by sandwich ELISA. The results (Table I⇓) show that each of these CTL lines secreted detectable quantities (generally in the pg/ml range) of each of these chemokines, although the levels varied widely. IL-16 was produced in far less amounts compared with IP-10, MIP-1α, or MIP-1β. No detectable chemokines were produced by APCs cultured alone (data not shown).

CD4⁺ T cell chemotaxis induced by products of CD8⁺PLP-specific CTL lines

Secretory products of PLP80–88- and PLP45–53-specific CTL lines were assayed for their ability to induce chemotaxis of CD4⁺ T cell lines GD22 (MBP87–106) and PLP40–60.1 and .2. Representative results are presented in Figure 1⇓.

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FIGURE 1.

Soluble products of PLP-specific CD8⁺ T cells are chemotactic for MBP- and PLP-specific CD4⁺ T cells. The PLP-specific CTL lines used for supernatant generation are: A, PLP80–88.1; B, PLP80–88.2; C, PLP45–53.1; D, PLP45–53.2; E, PLP80–88.3; F, PLP45–53.3; G, PLP45–53.4; H, PLP80–88.1; and I, PLP45–53.2. In A–D, the MBP87–106 CD4⁺ T cell line GD22 was used as the indicator cell for chemotaxis; in E–G, the PLP40–60 CD4⁺ T cell line PLP40–60.1 was used; and in H and I, PLP40–60.2 was used as the indicator cell line. Values on the y-axis represent the mean percentage of cell migration of triplicate determinations in one experiment induced by undiluted supernatants, and the error bars are the SD of these triplicates.

Each of the CD8⁺PLP-specific CTL lines produced substances that could induce migration of the CD4⁺MBP peptide-specific T cell line GD22 (Fig. 1⇑A–D) and the PLP40–60.1 (Fig. 1⇑E–G) and PLP40–60.2 (Fig. 1⇑, H and I) T cell lines. The activity present in undiluted supernatants induced cell migration of greater than 180% of control, with most CD8⁺PLP-specific CTL lines producing 300% cell migration. The chemotactic activity was dose dependent, as shown by the effect of diluting each of the supernatants (data not shown). These results demonstrate that CD8⁺PLP-specific CTL lines secrete factors that can induce chemotaxis of CD4⁺ MBP and PLP peptide-specific T cells.

Identification of CD8⁺ T cell products that are chemotactic for CD4⁺ T cells

Neutralizing antisera that are specific for IP-10, IL-16, MIP-1α, and MIP-1β were assayed for their ability to inhibit the chemotactic activity produced by representative CD8⁺ T cell lines. The neutralizing capacity of each of the antisera is provided in Materials and Methods. Each of the antisera was used in the chemotaxis assay at neutralizing concentrations that exceeded the concentration of the relevant chemokine in the supernatant. The results of three experiments are shown in Table II⇓. Little or no inhibition of chemotactic activity was produced by anti-MIP-1α or anti-MIP-1β. Anti-IL-16 produced 17 to 21% inhibition, while anti-IP-10 inhibited 36 to 59% of the chemotactic activity. These results demonstrate that the majority of the chemotactic activity for CD4⁺ T cells that is produced by the CD8⁺CTL line PLP80–88.3 is mediated by IP-10.

Similar analyses of the identity of the chemotactic agent present in the supernatant of CD8⁺CTL line PLP45–53.4 were performed. One representative experiment of three performed is shown in Figure 2⇓ using MBP-specific GD22 CD4⁺ T cells as indicator cells. The results are remarkably similar to the results obtained with the PLP80–88.3 CTL line: anti-IP-10 inhibited by 41%, followed by anti-IL-16 with 25%, followed by anti-MIP-1α and anti-MIP-1β with 14% and 13% inhibition, respectively. Again, these results indicate that the majority of the chemotactic activity for the CD4⁺ T cells produced by these CD8⁺ T cells is mediated by IP-10.

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FIGURE 2.

Ab neutralization of the chemotactic activity of soluble mediators secreted by PLP45–53.4. The supernatant from PLP45–53.4 was incubated with the indicated Abs. The indicator CD4⁺ T cell line GD22 specific for MBP87–106 was then added, and chemotactic activity was quantitated. Results are expressed as percentage of inhibition of cell migration. p values represent comparisons with percentage of cell migration in the absence of Abs.

The inability to attribute any significant chemotactic activity for the CD4⁺ T cells to either MIP-1α or MIP-1β was not due to a lack of expression of an appropriate cell surface chemokine receptor on these T cells, because they showed positive cell surface staining for chemokine receptor 5 using the 5C7 mAb (22) (data not shown).

Direct demonstration that IP-10 can induce chemotaxis of CD4⁺ myelin peptide-specific T cells

Purified human rIP-10 was assessed for its ability to induce chemotaxis of PLP40–60.1 CD4⁺ T cells. The results (Fig. 3⇓) demonstrate that rIP-10 induced detectable chemotactic activity of PLP40–60.1 T cells in the range of 100 to 10 ng/ml. Similar levels of percentage of cell migration were produced by undiluted supernatants from CD8⁺ T cell lines PLP80–88.3, PLP45–53.1, and PLP45–53.4 (Fig. 3⇓). Similar results were observed for induction of chemotactic activity by MBP-specific GD22 CD4⁺ T cells (data not shown). These results directly demonstrate that IP-10 alone can induce chemotaxis of CD4⁺ myelin peptide-specific T cell clones.

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FIGURE 3.

IP-10 can directly induce chemotaxis of CD4⁺ myelin peptide-specific T cells. PLP40.60.1 T cell line was tested in the chemotaxis assay with the indicated concentrations of human rIP-10 or the indicated undiluted supernatants.