Mechanisms of major histocompatibility complex class II-restricted processing and presentation of the V antigen of Yersinia pestis

Abstract

We mapped mouse CD4 T-cell epitopes located in three structurally distinct regions of the V antigen of Yersinia pestis. T-cell hybridomas specific for epitopes from each region were generated to study the mechanisms of processing and presentation of V antigen by bone-marrow-derived macrophages. All three epitopes required uptake and/or processing from V antigen as well as presentation to T cells by newly synthesized major histocompatibility complex (MHC) class II molecules over a time period of 3-4 hr. Sensitivity to inhibitors showed a dependence on low pH and cysteine, serine and metalloproteinase, but not aspartic proteinase, activity. The data indicate that immunodominant epitopes from all three structural regions of V antigen were presented preferentially by the classical MHC class II-restricted presentation pathway. The requirement for processing by the co-ordinated activity of several enzyme families is consistent with the buried location of the epitopes in each region of V antigen. Understanding the structure-function relationship of multiple immunodominant epitopes of candidate subunit vaccines is necessary to inform choice of adjuvants for vaccine delivery. In the case of V antigen, adjuvants designed to target it to lysosomes are likely to induce optimal responses to multiple protective T-cell epitopes.

Figure 1

Lymph node proliferation to peptides of V antigen. Groups of four or six BALB/c (a,c) or B10.D2 (b,d) mice were immunized with rV in adjuvant and popliteal lymph node proliferation was assayed 7 days later. Responses to 20 μg/ml rV and either (a,b) eleven V peptide pools, or (c,d) individual peptides from stimulatory pools. Peptides 9–11 are from pool 4, peptides 16–18 from pool 6, peptides 18–20 from pool 7, and peptides 21–23 from pool 8; the pools and peptides are defined in the Materials and methods section. B10.D2 mice were not assayed against individual peptides 18–20. Proliferation was measured by tritiated thymidine incorporation and the results are expressed as mean c.p.m. for all individual mice. All groups were compared to the no-antigen controls (0 on the x-axis) by unpaired Student’s t-test and when significantly different from the control P-values are shown as < 0·05 (+), < 0·01 (*), < 0·001 (**) and < 0·001 (***).

Figure 2

Structure of V antigen and location of H-2^d-restricted CD4 T-cell epitopes. Ribbon diagram of the structure of V created by DS ViewerPro 5·0 software (Accelrys Inc., Cambridge, UK) using the pdb file 1R6F to indicate the location of the immunodominant epitopes recognized by H-2^d CD4 T cells. The N-terminal amino acids Met1–His27 and C-terminal Thr323–Lys326 are not represented in the crystal structure.

Figure 3

Kinetics of uptake and presentation of rV to T cells. Macrophages were fixed in 1% paraformaldehyde either immediately before (prefixed), or 5 hr after (postfixed) exposure to 20 μg/ml rV or synthetic peptides and addition of the H-2^d-restricted T-cell hybridomas 2H1, 1E.10.2 and 1B4, specific for the epitopes within peptides 102–121 (a), 162–181 (b) or 212–231 (c), respectively. Alternatively, macrophages were incubated with rV or peptide for the times shown before fixation, washing and addition of the same T-cell hybridomas (d–f). After 24 hr interleukin-2 content of hybdridoma supernatants was measured in a biossay in which CTLL-2 proliferation was measured as tritiated thymidine incorporation and results expressed as mean c.p.m. ± SD.

Figure 4

Source of MHC class II for antigen presentation of rV to T cells. Macrophages were pretreated with (a–c) 1 μg/ml brefeldin A or (d–f) 20 μm cycloheximide for 3 hr before adding rV (20 μg/ml) or peptides (4 μg/ml) for a further 5 hr. Macrophages were then fixed, washed and assayed as described in the legend to Fig. 3 with H-2^d-restricted T-cell hybridomas 2H1, 1E.10.2 and 1B4, specific for the epitopes within peptides 102–121 (a, d), 162–181 (b, e) or 212–231 (c, f), respectively.

Figure 5

Effect of inhibitors of antigen processing of rV. (a–c) Macrophages were treated with 100 mm ammonium chloride or 4 µm monensin, both of which raise endosomal pH, or (d–f) with the broad specificity enzyme inhibitors, 2·5 μm E-64d, 350 μm pepstatin A, 400 μm 1,10-phenanthroline, 40 μm DCI. rV or peptides were added for 5 hr over the dose range shown before fixation and washing. The inhibitors at these doses did not affect presentation of 4 μg/ml of the relevant synthetic peptides (g–i). Macrophages were then assayed as described in the legend to Fig. 3 with T-cell hybridomas 2H1, 1B9 and 1B4, specific for epitopes within peptides 102–121 (a,d,g), 162–181(b,e,h) or 212–231 (c,f,i), respectively.