Extending the CD4(+) T-cell epitope specificity of the Th1 immune response to an antigen using a Salmonella enterica serovar typhimurium delivery vehicle

Abstract

We analyzed the CD4 T-cell immunodominance of the response to a model antigen (Ag), MalE, when delivered by an attenuated strain of Salmonella enterica serovar Typhimurium (SL3261*pMalE). Compared to purified MalE Ag administered with adjuvant, the mapping of the peptide-specific proliferative responses showed qualitative differences when we used the Salmonella vehicle. We observed the disappearance of one out of eight MalE peptides' T-cell reactivity upon SL3261*pMalE immunization, but this phenomenon was probably due to a low level of T-cell priming, since it could be overcome by further immunization. The most striking effect of SL3261*pMalE administration was the activation and stimulation of new MalE peptide-specific T-cell responses that were silent after administration of purified Ag with adjuvant. Ag presentation assays performed with MalE-specific T-cell hybridomas showed that infection of Ag-presenting cells by this intracellular attenuated bacterium did not affect the processing and presentation of the different MalE peptides by major histocompatibility complex (MHC) class II molecules and therefore did not account for immunodominance modulation. Thus, immunodominance of the T-cell response to microorganisms is governed not only by the frequency of the available T-cell repertoire or the processing steps in Ag-presenting cells that lead to MHC presentation but also by other parameters probably related to the infectious process and to the bacterial products. Our results indicate that, upon infection by a microorganism, the specificity of the T-cell response induced against its Ags can be much more effective than with purified Ags and that it cannot completely be mimicked by purified Ags administered with adjuvant.

FIG. 1

Mapping of the dominant T-cell determinants of the MalE protein in C57BL/6 mice. C57BL/6 mice were primed s.c. with 10 μg of MalE protein emulsified in CFA. Ten days later, LN cells were stimulated in vitro with overlapping 15-mer peptides (6.5 μM concentration) covering the entire MalE sequence in a single amino acid step, which is referred as the NH₂-terminal amino acid of each peptide. Proliferation was determined by ³H incorporation on day 4.

FIG. 2

Stimulation of MalE-specific T-cell hybridomas using SL3261*pMalE. (A) PEC were incubated for 2 h with either 100 μg of purified MalE protein per ml, 10⁷ SL3261*pMalE bacteria per ml, or 10⁷ control SL3261* bacteria per ml and were then fixed with glutaraldehyde. These APC were used to stimulate the different MalE-specific T-cell hybridomas with the indicated specificity. (B and C) PEC were first incubated with or without 10 μM CCB. Serial dilutions of purified MalE protein or SL3261*pMalE were then added for 2 h. Fixed PEC were then cultured with the KRM.E3 T-cell hybridoma. The IL-2 content in the 24-h supernatant was measured using the CTLL cell line.

FIG. 3

Analysis of the MalE-specific T-cell repertoire stimulated in vivo by SL3261*pMalE. C57BL/6 mice (two per group) were i.p. immunized on days 0 and 21 with 10⁶ SL3261*pMalE or control SL3261* bacteria. Spleen cells from each group were pooled and were stimulated in vitro with the indicated Ag. (A) Indicated MalE synthetic peptides were tested individually at 10 μM, purified MalE was tested at 0.25 μM, and SL3261* extract was tested at a dilution corresponding to 10⁶ bacteria/ml. (B) Series of 12 peptides were pooled (2 μM concentration of each peptide) and tested for reactivity. The peptide pools are referred to as the sequence encompassed by peptide series. Proliferation was determined by ³H incorporation on day 4. Asterisks indicate positive proliferative responses corresponding to those depicted in Fig. 1.

FIG. 4

Stimulation of p40–54-specific T-cell hybridoma by purified MalE or SL3261*pMalE. (A) The 53C1 T-cell hybridoma was stimulated by a 10-μM concentration of synthetic peptides corresponding to residues 35 to 60 from MalE in the presence of an I-A^b transfected L cell. (B) PEC were incubated for 2 h with SL3261*pMalE or SL3261*. After fixation these cells were used to stimulate the 53C1 T-cell hybridoma. (C) PEC were incubated with 10 μg of MalE per ml or with 10⁶ SL3261*pMalE bacteria per ml alone, with 10 μM BFA, or with 10 μM CCB. The IL-2 content in the 24-h supernatant was measured using the CTLL cell line.

FIG. 5

The crypticity of p40–54 following immunization with purified MalE in adjuvant is not due to inefficient processing in vivo. (A) Mice were s.c. immunized with 10 μg of MalE or p40–54 in CFA. Ten days later, LN cells were restimulated with serial dilutions of the p40–54 peptide. Proliferation was determined by ³H incorporation on day 4. (B, C, and D) Mice were s.c. immunized with MalE in adjuvant. Three days later, T-cell-depleted LN cells were directly used as APC to stimulate T-cell hybridomas specific for p40–54 (53C1) or p221–235 (ORMC7.9). (B) One hundred micrograms of MalE was administered in IFA or alum as indicated. (C and D) Indicated doses of MalE were injected together with IFA.

FIG. 6

Salmonella delivery of MalE does not modify the MHC presentation pattern of epitopes by Mφ and DC. PEC (A, B, and C) and DC (D, E, and F) were incubated for 2 and 3 h, respectively, with serial doses of SL3261*pMalE or purified MalE. After fixation these cells were used to stimulate the following T-cell hybridomas: ORM.C7.9 specific for p221–235 (A and D), 53C1 specific for p40–54 (B and E), or TRM.F2.1 specific for p262–276 (C and F). The IL-2 content in the 24-h supernatant was measured using the CTLL cell line.

FIG. 7

The route of immunization does not account for the modulation of the peptide-specific T-cell priming for purified MalE and MalE delivered by SL3261*. (A) C57BL/6 mice (two per group) were i.p. immunized on days 0 and 21 with 100 μg of MalE in alum or with 10⁶ SL3261*pMalE or control SL3261* bacteria. Spleen cells from each group were pooled and were stimulated in vitro with the indicated Ag and tested for proliferation on day 4. (B and C). Mice were intravenously immunized with a single dose of 10⁶ SL3261*pMalE or control SL3261* bacteria. Three (B) and four (C) weeks later, spleen cells from individual mice (two per group) were recovered and tested for IFN-γ-producing cells by ELISPOT in response to the indicated antigenic stimulation. Each data point corresponds to the mean of the number of positive CD4⁺ T cells per spleen obtained in two individual mice.

FIG. 8

Multiple immunizations with SL3261*pMalE enable p262–276-specific T-cell priming. A total of 10⁶ SL3261*pMalE (open symbols) or control SL3261* (closed symbols) bacteria were injected i.p. in mice (five per group) on day 0 (A), days 0 and 21 (B), and days 0, 21, and 42 (C). Three weeks after the last immunization, spleen cells from individual mice were stimulated in vitro with the indicated Ag. Proliferation was determined by ³H incorporation on day 4. Each data point corresponds to an individual mouse.

FIG. 9

In vitro-pulsed APC are able to prime T cells in vivo regardless of the Ag formulation. PEC were incubated for 4 h with 100 μg of MalE per ml alone, with 10 μg of lipopolysaccharide per ml, or with SL3261*pMalE or SL3261*. These cells were then i.p. injected in mice. Two weeks later, spleen cells were restimulated with MalE, p221–235, p40–54, or p262–276. Proliferation was determined by ³H incorporation on day 4.