Kevin B. Urdahl

The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation

Several subsets of Foxp3+ regulatory T cells (Treg cells) work in concert to maintain immune homeostasis. However, the molecular bases underlying the phenotypic and functional diversity of Treg cells remain obscure. We show that in response to interferon-γ, Foxp3+ Treg cells upregulated the T helper type 1 (TH1)-specifying transcription factor T-bet. T-bet promoted expression of the chemokine receptor CXCR3 on Treg cells, and T-bet+ Treg cells accumulated at sites of TH1 cell–mediated inflammation. Furthermore, T-bet expression was required for the homeostasis and function of Treg cells during type 1 inflammation. Thus, in a subset of CD4+ T cells, the activities of the transcription factors Foxp3 and T-bet are overlaid, which results in Treg cells with unique homeostatic and migratory properties optimized for the suppression of TH1 responses in vivo.

Expansion and function of Foxp3-expressing T regulatory cells during tuberculosis

Mycobacterium tuberculosis (Mtb) frequently establishes persistent infections that may be facilitated by mechanisms that dampen immunity. T regulatory (T reg) cells, a subset of CD4+ T cells that are essential for preventing autoimmunity, can also suppress antimicrobial immune responses. We use Foxp3-GFP mice to track the activity of T reg cells after aerosol infection with Mtb. We report that during tuberculosis, T reg cells proliferate in the pulmonary lymph nodes (pLNs), change their cell surface phenotype, and accumulate in the pLNs and lung at a rate parallel to the accumulation of effector T cells. In the Mtb-infected lung, T reg cells accumulate in high numbers in all sites where CD4+ T cells are found, including perivascular/peribronchiolar regions and within lymphoid aggregates of granulomas. To determine the role of T reg cells in the immune response to tuberculosis, we generated mixed bone marrow chimeric mice in which all cells capable of expressing Foxp3 expressed Thy1.1. When T reg cells were depleted by administration of anti-Thy1.1 before aerosol infection with Mtb, we observed ∼1 log less of colony-forming units of Mtb in the lungs. Thus, after aerosol infection, T reg cells proliferate and accumulate at sites of infection, and have the capacity to suppress immune responses that contribute to the control of Mtb.

Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids.

The evolutionary survival of Mycobacterium tuberculosis, the cause of human tuberculosis, depends on its ability to invade the host, replicate, and transmit infection. At its initial peripheral infection site in the distal lung airways, M. tuberculosis infects macrophages, which transport it to deeper tissues. How mycobacteria survive in these broadly microbicidal cells is an important question. Here we show in mice and zebrafish that M. tuberculosis, and its close pathogenic relative Mycobacterium marinum, preferentially recruit and infect permissive macrophages while evading microbicidal ones. This immune evasion is accomplished by using cell-surface-associated phthiocerol dimycoceroserate (PDIM) lipids to mask underlying pathogen-associated molecular patterns (PAMPs). In the absence of PDIM, these PAMPs signal a Toll-like receptor (TLR)-dependent recruitment of macrophages that produce microbicidal reactive nitrogen species. Concordantly, the related phenolic glycolipids (PGLs) promote the recruitment of permissive macrophages through a host chemokine receptor 2 (CCR2)-mediated pathway. Thus, we have identified coordinated roles for PDIM, known to be essential for mycobacterial virulence, and PGL, which (along with CCR2) is known to be associated with human tuberculosis. Our findings also suggest an explanation for the longstanding observation that M. tuberculosis initiates infection in the relatively sterile environment of the lower respiratory tract, rather than in the upper respiratory tract, where resident microflora and inhaled environmental microbes may continually recruit microbicidal macrophages through TLR-dependent signalling.

Positive selection of MHC class Ib-restricted CD8+ T cells on hematopoietic cells

Unlike conventional CD8+ T cells, major histocompatibility complex (MHC) class Ib–restricted CD8+ T cells show an activated phenotype in uninfected mice and respond rapidly to foreign invaders. The underlying factors that contribute to these differences are not well understood. We show here that the activated phenotype of MHC class Ib–restricted CD8+ T cells was partially acquired as a result of interactions in the thymus and reflected an increased capacity to be selected via interactions with MHC molecules on hematopoietic cells. Using bone marrow–chimeric mice, we have shown that MHC class Ib–restricted, but not MHC class Ia–restricted, CD8+ T cells specific for Listeria monocytogenes were efficiently selected when MHC class I was expressed only on hematopoietic cells. Thus, the distinct functional properties of MHC class Ib–restricted versus MHC class Ia–restricted CD8+ T cells may result, at least in part, from the different ways in which they are positively selected in the thymus.

Pathogen-specific Treg cells expand early during mycobacterium tuberculosis infection but are later eliminated in response to Interleukin-12.

Thymically derived Foxp3⁺ regulatory T (Treg) cells have a propensity to recognize self-peptide:MHC complexes, but their ability to respond to epitope-defined foreign antigens during infectious challenge has not been demonstrated. Here we show that pulmonary infection with Mycobacterium tuberculosis (Mtb), but not Listeria monocytogenes (Lm), induced robust lymph node expansion of a highly activated population of pathogen-specific Treg cells from the pre-existing pool of thymically derived Treg cells. These antigen-specific Treg cells peaked in numbers 3 weeks after infection but subsequently underwent selective elimination driven, in part, by interleukin-12-induced intrinsic expression of the Th1-cell-promoting transcription factor T-bet. Thus, the initial Mtb-induced inflammatory response promotes pathogen-specific Treg cell proliferation, but these cells are actively culled later, probably to prevent suppression during later stages of infection. These findings have important implications for the prevention and treatment of tuberculosis and other chronic diseases in which antigen-specific Treg cells restrict immunity.

ICOS and Bcl6-dependent pathways maintain a CD4 T cell population with memory-like properties during tuberculosis

Protective CD4 T cells specific for M. tuberculosis (Mtb) are maintained in the lungs during active Mtb infection. Similar to memory CD4 T cells, persistence of these Mtb-specific cells requires intrinsic expression of Bcl6 and ICOS.

CD8+ T Cells Accumulate in the Lungs of Mycobacterium tuberculosis-Infected Kb−/−Db−/− Mice, But Provide Minimal Protection

Recent studies have shown that MHC class I molecules play an important role in the protective immune response to Mycobacterium tuberculosis infection. Here we showed that mice deficient in MHC class Ia, but possessing MHC class Ib (Kb−/−Db−/− mice), were more susceptible to aerosol infection with M. tuberculosis than control mice, but less susceptible than mice that lack both MHC class Ia and Ib (β2m−/− mice). The susceptibility of Kb−/−Db−/− mice cannot be explained by the failure of CD8+ T cells (presumably MHC class Ib-restricted) to respond to the infection. Although CD8+ T cells were a relatively small population in uninfected Kb−/−Db−/− mice, most already expressed an activated phenotype. During infection, a large percentage of these cells further changed their cell surface phenotype, accumulated in the lungs at the site of infection, and were capable of rapidly producing IFN-γ following TCR stimulation. Histopathologic analysis showed widespread inflammation in the lungs of Kb−/−Db−/− mice, with a paucity of lymphocytic aggregates within poorly organized areas of granulomatous inflammation. A similar pattern of granuloma formation has previously been observed in other types of MHC class I-deficient mice, but not CD8α−/− mice. Thus, neither the presence of MHC class Ib molecules themselves, nor the activity of a population of nonclassical CD8+ effector cells, fully restored the deficit caused by the absence of MHC class Ia molecules, suggesting a unique role for MHC class Ia molecules in protective immunity against M. tuberculosis.

Foxp3(+) regulatory T cells in tuberculosis.

The immune response to Mycobacterium tuberculosis (Mtb) must be tightly regulated to mount a sufficient response to limit bacterial growth and dissemination while avoiding excessive inflammation that could damage host tissues. A wide variety of cell types, cell surface molecules, and cytokines are likely to contribute to this regulation, but recent studies have revealed that a subset of CD4 T cells expressing the transcription factor Foxp3, called regulatory T (reg) cells, play a critical role [1-3]. Although the first reports of T reg cells in tuberculosis (TB) occurred only recently (i.e., 2006) [4, 5], we have already gained many insights into their activity during TB. While it is likely that T reg cells do play some beneficial roles by preventing inflammation-mediated damage to host tissues during TB, this aspect of their function has not been well studied to date. What is clear, however, is that during the initial T cell response to Mtb infection, Mtb induces the expansions of T reg cells that delay the onset of adaptive immunity, suggesting that Mtb has hijacked T reg cell-mediated immune suppression to allow it to replicate unabated in the lung until T cells finally arrive [6]. In this chapter, we will first provide an overview of the delayed T cell response to Mtb and a brief introduction to regulatory T cells. We will then review what is known about T reg cells from observations in human populations, discuss mechanistic insights revealed in the mouse model, and speculate about the relevance of this understanding for future efforts to prevent and treat TB.

PCR and sequencing of independent genetic targets for the diagnosis of culture negative bacterial endocarditis

Molecular methods utilizing broad-range primers for 16S rDNA PCR and sequencing have been widely evaluated for their utility in culture negative diagnostic bacteriology. Difficulties in determining the incidence of false positive PCR results, especially in the absence of an equally sensitive confirmatory method however, have prevented wide clinical use of this sensitive technology. Here we report two cases of culture-negative endocarditis, in which PCR using 16S rDNA broad-range primers generated sequences specific for Bartonella spp. and Streptococcus oralis, respectively. To confirm these results, a second species- or genus-specific molecular target was chosen for each organism and detected in the split samples sequencially. Thus, molecular detection of a second species-specific target can be used to confirm PCR results generated from 16S rDNA broad-range primers and to control for potential false positive results due to environmental and amplicon carry-over contamination during specimen processing and testing in the laboratory.

Immune vulnerability of infants to tuberculosis.

One of the challenges faced by the infant immune system is learning to distinguish the myriad of foreign but nonthreatening antigens encountered from those expressed by true pathogens. This balance is reflected in the diminished production of proinflammatory cytokines by both innate and adaptive immune cells in the infant. A downside of this bias is that several factors critical for controlling Mycobacterium tuberculosis infection are significantly restricted in infants, including TNF, IL-1, and IL-12. Furthermore, infant T cells are inherently less capable of differentiating into IFN-γ-producing T cells. As a result, infected infants are 5–10 times more likely than adults to develop active tuberculosis (TB) and have higher rates of severe disseminated disease, including miliary TB and meningitis. Infant TB is a fundamentally different disease than TB in immune competent adults. Immunotherapeutics, therefore, should be specifically evaluated in infants before they are routinely employed to treat TB in this age group. Modalities aimed at reducing inflammation, which may be beneficial for adjunctive therapy of some forms of TB in older children and adults, may be of no benefit or even harmful in infants who manifest much less inflammatory disease.