Christopher C. Dascher

IL-10–producing T cells suppress immune responses in anergic tuberculosis patients

The lethality of Mycobacterium tuberculosis remains the highest among infectious organisms and is linked to inadequate immune response of the host. Containment and cure of tuberculosis requires an effective cell-mediated immune response, and the absence, during active tuberculosis infection, of delayed-type hypersensitivity (DTH) responses to mycobacterial antigens, defined as anergy, is associated with poor clinical outcome. To investigate the biochemical events associated with this anergy, we screened 206 patients with pulmonary tuberculosis and identified anergic patients by their lack of dermal reactivity to tuberculin purified protein derivative (PPD). In vitro stimulation of T cells with PPD induced production of IL-10, IFN-gamma, and proliferation in PPD(+) patients, whereas cells from anergic patients produced IL-10 but not IFN-gamma and failed to proliferate in response to this treatment. Moreover, in anergic patients IL-10-producing T cells were constitutively present, and T-cell receptor-mediated (TCR-mediated) stimulation resulted in defective phosphorylation of TCRzeta and defective activation of ZAP-70 and MAPK. These results show that T-cell anergy can be induced by antigen in vivo in the intact human host and provide new insights into mechanisms by which M. tuberculosis escapes immune surveillance.

Dissemination of Mycobacterium tuberculosis Is Influenced by Host Factors and Precedes the Initiation of T-Cell Immunity

ABSTRACT We report that dissemination of Mycobacterium tuberculosis in the mouse is under host control and precedes the initiation of T-cell immunity. Nine to eleven days after aerosol inoculation, M. tuberculosis disseminates to the pulmonary lymph nodes (LN), where M. tuberculosis-specific T cells are detected 2 to 3 days thereafter. This indicates that the initial spread of bacteria occurs via lymphatic drainage and that the acquired T-cell immune response is generated in the draining LN. Dissemination to peripheral sites, such as the spleen and the liver, occurs 11 to 14 days postinfection and is followed by the appearance of M. tuberculosis-specific T cells in the lung and the spleen. In all cases studied, dissemination to the LN or the spleen preceded activation of M. tuberculosis-specific T cells in that organ. Interestingly, bacteria disseminate earlier from the lungs of resistant C57BL/6 mice than from the lungs of susceptible C3H mice, and consequently, C57BL/6 mice generate an immune response to M. tuberculosis sooner than C3H mice generate an immune response. Thus, instead of spreading infection, early dissemination of M. tuberculosis may aid in the initiation of an appropriate and timely immune response. We hypothesize that this early initiation of immunity following inoculation with M. tuberculosis may contribute to the superior resistance of C57BL/6 mice.

Detection of prokaryotic mRNA signifies microbial viability and promotes immunity

Live vaccines have long been known to trigger far more vigorous immune responses than their killed counterparts. This has been attributed to the ability of live microorganisms to replicate and express specialized virulence factors that facilitate invasion and infection of their hosts. However, protective immunization can often be achieved with a single injection of live, but not dead, attenuated microorganisms stripped of their virulence factors. Pathogen-associated molecular patterns (PAMPs), which are detected by the immune system, are present in both live and killed vaccines, indicating that certain poorly characterized aspects of live microorganisms, not incorporated in dead vaccines, are particularly effective at inducing protective immunity. Here we show that the mammalian innate immune system can directly sense microbial viability through detection of a special class of viability-associated PAMPs (vita-PAMPs). We identify prokaryotic messenger RNA as a vita-PAMP present only in viable bacteria, the recognition of which elicits a unique innate response and a robust adaptive antibody response. Notably, the innate response evoked by viability and prokaryotic mRNA was thus far considered to be reserved for pathogenic bacteria, but we show that even non-pathogenic bacteria in sterile tissues can trigger similar responses, provided that they are alive. Thus, the immune system actively gauges the infectious risk by searching PAMPs for signatures of microbial life and thus infectivity. Detection of vita-PAMPs triggers a state of alert not warranted for dead bacteria. Vaccine formulations that incorporate vita-PAMPs could thus combine the superior protection of live vaccines with the safety of dead vaccines.

Cadherin-11 Provides Specific Cellular Adhesion between Fibroblast-like Synoviocytes

Cadherins are integral membrane proteins expressed in tissue-restricted patterns that mediate homophilic intercellular adhesion. During development, they orchestrate tissue morphogenesis and, in the adult, they determine tissue integrity and architecture. The synovial lining is a condensation of fibroblast-like synoviocytes (FLS) and macrophages one to three cells thick. These cells are embedded within the extracellular matrix, but the structure is neither an epithelium nor an endothelium. Previously, the basis for organization of the synovium into a tissue was unknown. Here, we cloned cadherin-11 from human rheumatoid arthritis (RA)-derived FLS. We developed L cell transfectants expressing cadherin-11, cadherin-11 fusion proteins, and anti–cadherin-11 mAb. Cadherin-11 was found to be expressed mainly in the synovial lining by immunohistologic staining of human synovium. FLS adhered to cadherin-11–Fc, and transfection of cadherin-11 conferred the formation of tissue-like sheets and lining-like structures upon fibroblasts in vitro. These findings support a key role for cadherin-11 in the specific adhesion of FLS and in synovial tissue organization and behavior in health and RA.

Protection Elicited by a Double Leucine and Pantothenate Auxotroph of Mycobacterium tuberculosis in Guinea Pigs

ABSTRACT We developed a live, fully attenuated Mycobacterium tuberculosis vaccine candidate strain with two independent attenuating auxotrophic mutations in leucine and pantothenate biosynthesis. The ΔleuD ΔpanCD double auxotroph is fully attenuated in the SCID mouse model and highly immunogenic and protective in the extremely sensitive guinea pig tuberculosis model, reducing both bacterial burden and disease pathology.

Mycobacterium tuberculosis Regulates CD1 Antigen Presentation Pathways through TLR-2

Mycobacterium tuberculosis remains a major pathogen of worldwide importance, which releases lipid Ags that are presented to human T cells during the course of tuberculosis infections. Here we report that cellular infection with live M. tuberculosis or exposure to mycobacterial cell wall products converted CD1− myeloid precursors into competent APCs that expressed group 1 CD1 proteins (CD1a, CD1b, and CD1c). The appearance of group 1 CD1 proteins at the surface of infected or activated cells occurred via transcriptional regulation, and new CD1 protein synthesis and was accompanied by down-regulation of CD1d transcripts and protein. Isolation of CD1-inducing factors from M. tuberculosis using normal phase chromatography, as well as the use of purified natural and synthetic compounds, showed that this process involved polar lipids that signaled through TLR-2, and we found that TLR-2 was necessary for the up-regulation of CD1 protein expression. Thus, mycobacterial cell wall lipids provide two distinct signals for the activation of lipid-reactive T cells: lipid Ags that activate T cell receptors and lipid adjuvants that activate APCs through TLR-2. These dual activation signals may represent a system for selectively promoting the presentation of exogenous foreign lipids by those myeloid APCs, which come into direct contact with pathogens.

Transcription Regulation by the Mycobacterium tuberculosis Alternative Sigma Factor SigD and Its Role in Virulence

Mycobacterium tuberculosis, an obligate mammalian pathogen, adapts to its host during the course of infection via the regulation of gene expression. Of the regulators of transcription that play a role in this response, several alternative sigma factors of M. tuberculosis have been shown to control gene expression in response to stresses, and some of these are required for virulence or persistence in vivo. For this study, we examined the role of the alternative sigma factor SigD in M. tuberculosis gene expression and virulence. Using microarray analysis, we identified several genes whose expression was altered in a strain with a sigD deletion. A small number of these genes, including sigD itself, the gene encoding the autocrine growth factor RpfC, and a gene of unknown function, Rv1815, appear to be directly regulated by this sigma factor. By identifying the in vivo promoters of these genes, we have determined a consensus promoter sequence that is putatively recognized by SigD. The expression of several genes encoding PE-PGRS proteins, part of a large family of related genes of unknown function, was significantly increased in the sigD mutant. We found that the expression of sigD is stable throughout log phase and stationary phase but that it declines rapidly with oxygen depletion. In a mouse infection model, the sigD mutant strain was attenuated, with differences in survival and the inflammatory response in the lung between mice infected with the mutant and those infected with the wild type.

Evolutionary constraints on CD1 structure: insights from comparative genomic analysis

Abstract It is well established that the CD1 family of proteins present lipid antigens to T cells. However, in vivo studies of CD1 function have been carried out almost exclusively using mouse models, despite the fact that mice lack most of the CD1 genes that are found in humans and other mammals. To understand why there is a difference in the number of CD1 genes observed between humans and mice, we used comparative genomic analysis to examine differences at the CD1 locus. This analysis reveals a significant genomic deletion in mice that provides a probable explanation for the disparity. Furthermore, we propose a hypothesis in which functionally convergent evolution could have a role in at least partially compensating for the loss of certain CD1 isoforms during the course of mammalian evolution.

Estradiol Acts Directly on Bone Marrow Myeloid Progenitors to Differentially Regulate GM-CSF or Flt3 Ligand-Mediated Dendritic Cell Differentiation

Estrogen receptor (ER) ligands modulate hemopoiesis and immunity in the normal state, during autoimmunity, and after infection or trauma. Dendritic cells (DC) are critical for initiation of innate and adaptive immune responses. We demonstrate, using cytokine-driven culture models of DC differentiation, that 17-β-estradiol exerts opposing effects on differentiation mediated by GM-CSF and Flt3 ligand, the two cytokines that regulate DC differentiation in vivo. We also show that estradiol acts on the same highly purified Flt3+ myeloid progenitors (MP) to differentially regulate the DC differentiation in each model. In GM-CSF-supplemented cultures initiated from MP, physiological amounts of estradiol promoted differentiation of Langerhans-like DC. Conversely, in Flt3 ligand-supplemented cultures initiated from the same MP, estradiol inhibited cell survival in a dose-dependent manner, thereby decreasing the yield of plasmacytoid and conventional myeloid and lymphoid DC. Experiments with bone marrow cells from ER-deficient mice and the ER antagonist ICI182,780 showed that estradiol acted primarily via ERα to regulate DC differentiation. Thus, depending on the cytokine environment, pathways of ER signaling and cytokine receptor signaling can differentially interact in the same Flt3+ MP to regulate DC development. Because the Flt3 ligand-mediated differentiation pathway is important during homeostasis, and GM-CSF-mediated pathways are increased by inflammation, our data suggest that endogenous or pharmacological ER ligands may differentially affect DC development during homeostasis and disease, with consequent effects on DC-mediated immunity.

CD1-restricted adaptive immune responses to Mycobacteria in human group 1 CD1 transgenic mice

Group 1 CD1 (CD1a, CD1b, and CD1c)–restricted T cells recognize mycobacterial lipid antigens and are found at higher frequencies in Mycobacterium tuberculosis (Mtb)–infected individuals. However, their role and dynamics during infection remain unknown because of the lack of a suitable small animal model. We have generated human group 1 CD1 transgenic (hCD1Tg) mice that express all three human group 1 CD1 isoforms and support the development of group 1 CD1–restricted T cells with diverse T cell receptor usage. Both mycobacterial infection and immunization with Mtb lipids elicit group 1 CD1–restricted Mtb lipid–specific T cell responses in hCD1Tg mice. In contrast to CD1d-restricted NKT cells, which rapidly respond to initial stimulation but exhibit anergy upon reexposure, group 1 CD1–restricted T cells exhibit delayed primary responses and more rapid secondary responses, similar to conventional T cells. Collectively, our data demonstrate that group 1 CD1–restricted T cells participate in adaptive immune responses upon mycobacterial infection and could serve as targets for the development of novel Mtb vaccines.