Induction of an antigen-specific, CD1-restricted cytotoxic T lymphocyte response In vivo

Abstract

The majority of T cell responses are restricted to peptide antigens bound by polymorphic major histocompatibility complex (MHC) molecules. However, peptide antigens can be presented to T cells by murine non-MHC-encoded CD1d (mCD1) molecules, and human T cell lines specific for nonpeptide antigens presented on CD1 isoforms have been identified. It is shown here that antigen-specific, mCD1-restricted lymphocytes can be generated in vivo by immunizing mice with a combination of plasmids encoding chicken ovalbumin, murine CD1d, and costimulatory molecules. Splenocytes from immunized mice have CD1d-restricted, MHC- unrestricted, ovalbumin-specific cytolytic activity that can be inhibited by anti-CD1 antibodies as well as a competing CD1-binding peptide. These results suggest a physiologic role for murine CD1d to present exogenous protein antigens.

Figure 1

Recognition of ovalbumin by CD1d1-restricted CTL. Three mice per group were immunized with 50 μg of each plasmid at days 0 and 7. 2 wk after the first injection, mice were killed and restimulated in vitro with CD1d1-transfected EL4 cells pulsed with ovalbumin protein for 5 d in the presence of 50 IU rIL-2. The specificity of the resulting pooled CTL was determined by measuring the lysis of CD1d1-transfected RMAS cells incubated with ovalbumin in triplicate. (A) Immunizing mice with a combination of plasmids enhances the CD1-restricted CTL response. Mice were immunized with plasmids encoding ovalbumin, mCD1d1, and B7.1 (diamonds); ovalbumin, mCD1d1, and B7.2 (crosses); ovalbumin (circles); ovalbumin and mCD1d1 (squares); or mCD1d1 (triangles). The values plotted are average protein-specific lysis − background lysis. (B) CTL are specific for CD1d1 and ovalbumin. The splenocytes from the three mice injected with plasmids encoding ovalbumin, mCD1d1, and B7.1 were tested for their ability to lyse CD1-transfected RMAS cells pulsed with ovalbumin (triangles), CD1-transfected RMAS (squares), or RMAS cells pulsed with ovalbumin (circles) in triplicate. The values plotted represent the average lysis for three mice. These experiments were repeated twice.

Figure 2

Antibody inhibition of CD1-restricted CTL lysis. Four C57BL/6 mice were immunized with plasmids encoding OVA, mCD1d1, and B7.1 as in Fig. 1. Spleens were harvested after 2 wk and restimulated in vitro for 5 d with CD1d1 transfected RMAS cells pulsed with ovalbumin protein for 5 d with rIL-2. The splenocytes were pooled and assayed for CTL activity in duplicate. The percentage of maximum lysis relative to the value in the presence of targets and antigen alone ± SEM is shown. This experiment was repeated five times.

Figure 3

Peptide inhibition of ovalbumin-specific, CD1-restricted CTL lysis. T cell lines from mice injected with plasmids encoding OVA, mCD1d1, and B7.1 were assayed for cytolytic activity. CD1⁺ RMAS targets incubated with effector cells in triplicate wells were either unpulsed, pulsed for 2 h with 4 μg/ml of p99, pulsed with 200 μg/ml of ovalbumin protein for 1 h, washed, and then pulsed with 12 μg/ml of p99 for 1 h, or pulsed with ovalbumin protein only. The percentage of lysis ± SEM is shown at an E/T ratio of 30:1. This experiment was repeated twice.

Figure 4

Ovalbumin-specific, CD1-restricted CTL lysis of allogeneic targets. 10 C57BL/6 mice (H-2^b) were immunized with plasmids encoding OVA, mCD1d1, and B7.1 (circles), and four mice were immunized with mCD1d1 and B7.1 (squares) on days 0 and 10. 2 wk after the first injection, mice were killed and restimulated in vitro with CD1d1-transfected RMAS cells pulsed with ovalbumin protein for 5 d in the presence of 50 IU/ ml rIL-2. CD1⁺ RMAS or CD1⁺ P815 targets were incubated in the presence or absence of ovalbumin protein (100 μg/ ml) for 1 h and washed three times before being plated with effector cells in triplicate wells. The specific lysis was determined by measuring the lysis of CD1⁺ RMAS cells pulsed with ovalbumin minus unpulsed transfectants (A) or CD1⁺ P815 cells pulsed with ovalbumin minus unpulsed transfectants (B). The values plotted represent the average lysis ± SEM. This experiment was repeated twice.