IL-21-dependent expansion of memory-like NK cells enhances protective immune responses against Mycobacterium tuberculosis.

Natural killer (NK) cells are traditionally considered as innate cells, but recent studies suggest that NK cells can distinguish antigens, and that memory NK cells expand and protect against viral pathogens. Limited information is available about the mechanisms involved in memory-like NK cell expansion, and their role in bacterial infections and vaccine-induced protective immune responses. In the current study, using a mouse model of tuberculosis (TB) infection, we found that interferon-gamma producing CD3-NKp46+CD27+KLRG1+ memory-like NK cells develop during Bacille Calmette-Guérin vaccination, expand, and provide protection against challenge with Mycobacterium tuberculosis (M. tb). Using antibodies, short interfering RNA and gene-deleted mice, we found that expansion of memory-like NK cells depends on interleukin 21 (IL-21). NKp46+CD27+KLRG1+ NK cells expanded in healthy individuals with latent TB infection in an IL-21-dependent manner. Our study provides first evidence that memory-like NK cells survive long term, expansion depends on IL-21, and involved in vaccine-induced protective immunity against a bacterial pathogen.

Listeria monocytogenes infection overcomes the requirement for CD40 ligand in exogenous antigen presentation to CD8(+) T cells.

In vivo priming of CD8(+) T lymphocytes against exogenously processed model Ags requires CD4(+) T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4(+) T cells and CD40, which is present on professional APC such as dendritic cells (DCs). To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8(+) T cell priming against an exogenously processed, nonsecreted bacterial Ag. CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8(+) T cell cross-priming against an exogenous model Ag, OVA. In contrast, MR-1 treatment did not interfere with CD8(+) T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes. Memory and secondary responses of CD8(+) T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment. When MR-1-treated mice were concurrently immunized with L. monocytogenes and OVA-loaded splenocytes, cross-priming of OVA-specific naive CD8(+) T cells occurred. No significant decline in cross-priming against OVA was measured when either TNF or IFN-gamma was neutralized in L. monocytogenes-infected animals, demonstrating that multiple signals exist to overcome CD40L blockade of CD8(+) T cell cross-priming during bacterial infection. These data support a model in which DCs can be stimulated in vivo through signals other than CD40, becoming APC that can effectively stimulate CD8(+) T cell responses against exogenous Ags during infection.

Identification of Listeria monocytogenes in vivo-induced genes by fluorescence-activated cell sorting.

Listeria monocytogenes is a gram-positive, intracellular, food-borne pathogen capable of causing severe infections in immunocompromised or pregnant individuals, as well as numerous animal species. Genetic analysis of Listeria pathogenesis has identified several genes which are crucial for virulence. The transcription of most of these genes has been shown to be induced upon entry of Listeria into the host cell. To identify additional genes that are induced in vivo and may be required for L. monocytogenes pathogenesis, a fluorescence-activated cell-sorting technique was initiated. Random fragments of the L. monocytogenes chromosome were cloned into a plasmid carrying a promoterless green fluorescent protein (GFP) gene, and the plasmids were transformed into the L. monocytogenes actA mutant DP-L1942. Fluorescence-activated cell sorting (FACS) was used to isolate L. monocytogenes clones that exhibited increased GFP expression within macrophage-like J774 cells but had relatively low levels of GFP expression when the bacteria were extracellular. Using this strategy, several genes were identified, including actA, that exhibited such an expression profile. In-frame deletions of two of these genes, one encoding the putative L. monocytogenes uracil DNA glycosylase (ung) and one encoding a protein with homology to the Bacillus subtilis YhdP hemolysin-like protein, were constructed and introduced into the chromosome of wild-type L. monocytogenes 10403s. The L. monocytogenes 10403s ung deletion mutant was not attenuated for virulence in mice, while the yhdP mutant exhibited a three- to sevenfold reduction in virulence.

Regulation of antigen-specific CD8+ T cell homeostasis by perforin and interferon-gamma.

T cell memory depends on factors that regulate expansion and death of these cells after antigenic stimulation. Mice deficient in perforin and interferon-gamma (IFN-gamma) exhibited increased expansion, altered immunodominance, and decreased death of antigen-specific CD8+ T cells after infection with an attenuated strain of Listeria monocytogenes, which was cleared from these mice. Expansion of CD8+ T cells was controlled by perforin, whereas IFN-gamma regulated immunodominance and the death phase. Thus, perforin and IFN-gamma regulate distinct elements of CD8+ T cell homeostasis independently of their role as antimicrobial effector molecules.

CD8+ T cell effector mechanisms in resistance to infection.

Based on T cell subset depletion studies and the analysis of gene knockout mice, it is evident that CD8(+) T cells contribute to resistance against intracellular infections with certain viral, protozoan, and bacterial pathogens. Although they are known primarily for their capacity to kill infected cells, CD8(+) T cells elaborate a variety of effector mechanisms with the potential to defend against infection. Microbes use multiple strategies to cause infection, and the nature of the pathogenhost interaction may determine which CD8(+) T cell effector mechanisms are required for immunity. In this review, we summarize our current understanding of the effector functions used by CD8(+) T cells in resistance to pathogens. Analyses of mice deficient in perforin and/or Fas demonstrate that cytolysis is critical for immunity against some, but not all, infections and also reveal the contribution of cytolysis to the pathogenesis of disease. The role of CD8(+) T cell-derived cytokines in resistance to infection has been analyzed by systemic treatment with neutralizing antibodies and cytokine gene knockout mice. These studies are complicated by the fact that few, if any, cytokines are uniquely produced by CD8(+) T cells. Thus, the requirement for CD8(+) T cell- derived cytokines in resistance against most pathogens remains to be defined. Finally, recent studies of human CD8(+) T cells reveal the potential for novel effector mechanisms in resistance to infection.

CD8(+) T-cell priming against a nonsecreted Listeria monocytogenes antigen is independent of the antimicrobial activities of gamma interferon.

Sublethal infection of mice with recombinant Listeria monocytogenes expressing a model epitope in either secreted or nonsecreted form results in similar CD8(+) T-cell priming. Since nonsecreted bacterial proteins have no obvious access to the endogenous major histocompatibility complex (MHC) class I presentation pathway, presentation of these antigens requires destruction of the bacterium to reveal the nonsecreted molecules to an exogenous MHC class I presentation pathway. Gamma interferon (IFN-gamma), a cytokine made by multiple cell types in response to L. monocytogenes infection, could be required for exogenous presentation of nonsecreted bacterial antigens via its capacity to upregulate the expression of molecules involved in antigen presentation, its capacity to activate macrophages to kill bacteria to expose nonsecreted molecules or both. IFN-gamma knockout (KO) mice were used to address the requirement for IFN-gamma in CD8(+) T-cell priming against (i) a model exogenous antigen and (ii) secreted and nonsecreted L. monocytogenes antigens. We demonstrate that IFN-gamma KO mice are capable of cross-presenting the model exogenous antigen ovalbumin to prime CD8(+) T-cell responses that are only slightly weaker than that in wild-type (WT) mice. Despite their extreme susceptibility to primary L. monocytogenes infection, previously immunized and naive IFN-gamma KO mice were able to generate CD8(+) T-cell responses against both secreted and nonsecreted L. monocytogenes antigens which were similar to responses of WT mice. Interestingly, IFN-gamma KO mice were as capable as WT mice in mediating the characteristic drop in bacterial load in the liver at 4 h postinfection, although the IFN-gamma KO mice have exacerbated bacterial loads as early as 24 h postinfection. These results demonstrate that the regulatory functions of IFN-gamma are not required for priming of CD8(+) T cells by cross-presentation of a model exogenous antigen or in response to a nonsecreted L. monocytogenes antigen. In addition, the capacity of IFN-gamma to activate the microbicidal activities of macrophages is not required for the very early innate immune response to L. monocytogenes or priming of CD8(+) T cells against a nonsecreted bacterial antigen.