Cutting Edge: Protective Cell-Mediated Immunity to Listeria monocytogenes in the Absence of Myeloid Differentiation Factor 88

Abstract

In addition to their role in triggering innate immune responses, Toll-like receptors are proposed to play a key role in linking the innate and adaptive arms of the immune response. The majority of cellular responses downstream of Toll-like receptors are mediated through the adapter molecule myeloid differentiation factor 88 (MyD88), and mice with a targeted deletion of MyD88 are highly susceptible to bacterial infections, including primary infection with Listeria monocytogenes (LM). In contrast, herein we demonstrate that MyD88-deficient mice have only a modest impairment in their LM-specific CD4 T cell response, and no impairment in their CD8 T cell response following infection with ActA-deficient LM. Furthermore, CD8 T cells from immunized MyD88-deficient mice protected naive recipient mice following adoptive splenocyte transfer, and immunized MyD88-deficient mice were protected from infection with wild-type LM. These results indicate that adaptive immune responses can be generated and provide protective immunity in the absence of MyD88.

Induction of Protective Immunity to Listeria monocytogenes in Neonates

“…⌬actA-Lm-OVA was constructed from Lm-OVA by cloning ϳ500 bp fragments of the actA gene into the HindIII/KpnI sites of the temperaturesensitive plasmid pKSV7 using the following primers: upstream flanking region, forward primer 5Ј-aagcttgcagcgaccgatagcgaag-3Ј, reverse primer 5Ј-gaattccgctgcgctatccgatgg-3Ј, downstream flanking region, forward primer 5Ј-gaattcgttaagtccaaaggtatcg-3Ј, reverse primer 5Ј-ggtacctaaagagaa cacgccaatag-3Ј (underlined sequences indicate introduced restriction sites). All strains were grown as described previously (13). In brief, Lm were grown to mid-log phase (OD 600 , 0.1) at 37°C, diluted in 100 l of saline, and injected i.p.…”

“…The concentration of IL-4 and IL-13 in the supernatants of splenocyte cultures was determined 72 h after peptide stimulation by ELISA using reagents from R&D Systems as previously described (13).…”

“…The ⌬actA strain of Lm carries an in-frame deletion in the actA gene (⌬actA-Lm), is unable to spread from cell-cell, and displays a three order of magnitude decrease in virulence without interfering with immunogenicity (10,14). We and others have shown that ⌬actA-Lm was safe in MyD88-deficient or IFN-␥-deficient adult mice, which, similar to neonates, are highly susceptible to Lm infection (13,(15)(16)(17). We found that neonatal mice survived high-dose infection with ⌬actA-Lm without any sign of disease.…”

IL-12 and Type-I IFN Synergize for IFN-γ Production by CD4 T Cells, Whereas Neither Are Required for IFN-γ Production by CD8 T Cells afterListeria monocytogenesInfection

“…Within an infected cell, unipolar expression of ActA by Lm facilitates and is required for bacterium to recruit host cell actin into organized tails, allowing for intracellular spread leading to a productive infection (25). We and others have demonstrated that infection with the ⌬actA Lm mutant is able to prime Lm-specific CD8 and CD4 T cells, and yet is nonlethal even at relatively high inocula (up to 10 6 CFUs) in mice deficient in components of innate immunity such as MyD88, IFN-␥, or TNF-␣ receptor normally essential for protection from WT Lm infection (20,26,27). To verify that the absence of either IL-12 or IFN-IR signaling does not significantly alter the Lm load following infection with Lm-OVA ⌬actA, we examined the number of bacteria in the spleen of IFN-IR Ϫ/Ϫ , IL-12P40 Ϫ/Ϫ , and control mice following infection with 10 6 CFUs of Lm-OVA ⌬actA (Fig.…”

“…For in vitro culture, splenocytes were plated into 96-well round-bottom plates (5 ϫ 10 6 cells/ ml), and either stained directly with tetramer, or stimulated with the indicated peptides or heat-killed Lm for 5 h (intracellular cytokine staining) or 72 h (culture supernatants), as described (8,20). Heat-killed Lm was prepared by washing and resuspending Lm in log-phase growth in PBS and incubating at 65°C for 30 min.…”