ML1419c peptide immunization induces Mycobacterium leprae-specific HLA-A*0201-restricted CTL in vivo with potential to kill live mycobacteria

Abstract

MHC class I-restricted CD8(+) T cells play an important role in protective immunity against mycobacteria. Previously, we showed that p113-121, derived from Mycobacterium leprae protein ML1419c, induced significant IFN-γ production by CD8(+) T cells in 90% of paucibacillary leprosy patients and in 80% of multibacillary patients' contacts, demonstrating induction of M. leprae-specific CD8(+) T cell immunity. In this work, we studied the in vivo role and functional profile of ML1419c p113-121-induced T cells in HLA-A*0201 transgenic mice. Immunization with 9mer or 30mer covering the p113-121 sequence combined with TLR9 agonist CpG induced HLA-A*0201-restricted, M. leprae-specific CD8(+) T cells as visualized by p113-121/HLA-A*0201 tetramers. Most CD8(+) T cells produced IFN-γ, but distinct IFN-γ(+)/TNF-α(+) populations were detected simultaneously with significant secretion of CXCL10/IFN-γ-induced protein 10, CXCL9/MIG, and VEGF. Strikingly, peptide immunization also induced high ML1419c-specific IgG levels, strongly suggesting that peptide-specific CD8(+) T cells provide help to B cells in vivo, as CD4(+) T cells were undetectable. An additional important characteristic of p113-121-specific CD8(+) T cells was their capacity for in vivo killing of p113-121-labeled, HLA-A*0201(+) splenocytes. The cytotoxic function of p113-121/HLA-A*0201-specific CD8(+) T cells extended into direct killing of splenocytes infected with live Mycobacterium smegmatis expressing ML1419c: both 9mer and 30mer induced CD8(+) T cells that reduced the number of ML1419c-expressing mycobacteria by 95%, whereas no reduction occurred using wild-type M. smegmatis. These data, combined with previous observations in Brazilian cohorts, show that ML1419c p113-121 induces potent CD8(+) T cells that provide protective immunity against M. leprae and B cell help for induction of specific IgG, suggesting its potential use in diagnostics and as a subunit (vaccine) for M. leprae infection.

Figure 1

Figure 1A: IFN-γ secretion. IFN-γ secretion was analyzed after 5 days in vitro stimulation of splenocytes with ML1419c p113-121, M. leprae sonicate or mitogen (conA). HLA-A2tg mice were unimmunized (upper left panel) or immunized with CpG alone (upper right panel), ML1419c p113-121 (9mer/ CpG; lower left panel) or ML1419c p100-129 (30mer/ CpG; lower right panel). The HLA-A*0201-restricted epitope Rv1886 (M. tuberculosis Ag85B) p143-152 (25) was used as a control for ML1419c-specificity. All test groups included five mice. All mice were separately analyzed. Results are shown for one animal and are representative for each test group. Figure 1B: Intracellular IFN-γ production by CD8⁺ T-cells after ML1419c p113-121 immunization of HLA-A2tg mice. Mice were immunized twice with CpG alone (upper left), ML1419c p113-121 (9mer/ CpG; upper right), p108-122 (15mer/ CpG; lower left) or p100-129 (30mer/ CpG; lower right). Splenocytes were stimulated in vitro with the same peptides used for immunization. After 7 days, cells were incubated with medium or fresh peptide for 1h before addition of brefeldin A and analysis for intracellular IFN-γ production. Figure 1C: Frequency of polyfunctional CD8⁺ T-cells. Percentage of CD8⁺ T-cells in HLA-A2tg mice, producing combinations of IFN-γ, TNF-α or IL-2 after in vitro stimulation with peptide (see 1B). Mice were immunized with ML1419c p113-121 (9mer/ CpG). The total number of CD8⁺ T-cells analysed in immunized mice was 56,000. Slices in pie chart represent the proportion of single, double or triple positive CD8⁺ T-cells for each antigen. Only CD8⁺ populations of > 5 × 10⁴ events were analyzed. In naïve mice the number of CD8⁺ T-cells producing cytokines was less than 5 × 10⁴c.

Figure 2. Tetramer staining

Splenocytes of unimmunized (A, C) or ML1419c p113-121-immunized (B, D) HLA- A2 tg mice were stained with PE-conjugated anti-CD8 and APC-conjugated HLA-A*0201/ml1419c p113-121 tetramer (HLA-A2/p113 TM) directly ex vivo (A, B) or after 1 week in vitro restimulation with ML1419c p113-121 (C, D).

Figure 3. Quantification of serum antibodies to ML1419c

Following immunization of HLA-A2tg mice with CpG alone (A) or with ML1419c p113-121/ CpG (B) antibody titers (OD₄₅₀) against ML1419c p113-121 (▼), ML1419c p100-129 (*) or ML1419c protein (●) were determined by ELISA. As a control affinity for BSA (0.4% in PBS) alone (■) is shown. Serum dilutions are shown on the x-axis. All test groups included five mice. All mice were separately analyzed. Results are shown for one animal and are representative for each test group.

Figure 4. In vivo cytotoxicity

CTL response in HLA-A2tg mice against ML1419c p113-121 as detected by lysis of ML1419c p113-121-pulsed, CFSE^high labeled syngeneic target cells using flow cytometry. The y-axis indicates the number of cells and the x-axis the CFSE intensity. The figure shows one representative CpG-immunized animal (A; n = 5), three representative animals from the groups immunized with ML1419c p113-121/CpG (B; n = 5) and ML1419c p100-129/CpG (C; n =5). Results shown are representative for at least four separate experiments. Correlation of ML1419c p113-121-specific in vivo lysis in various experiments with the percentage of HLA-A2/p113 TM⁺ CD8⁺ T-cells directly ex vivo (D) or after in vitro peptide restimulation (E).

Figure 5. Determination of colony forming units (CFU) of recombinant M. smegmatis expressing ML1419c antigen

CFU were determined for M. smegmatis expressing ML1419C (pVV:ML1419) (A) or M. smegmatis with empty vector (pVV16) (B) after incubation with splenocytes derived from HLA-A2tg mice immunized with CpG (white bar), ML1419c p113-121/CpG (black bar) or ML1419c p100-129/CpG (grey bar).