Intrinsic immunogenicity of liposomes for tuberculosis vaccines: Effect of cationic lipid and cholesterol.

Tuberculosis (TB) is still among the deadliest infectious diseases, hence there is a pressing need for more effective TB vaccines. Cationic liposome subunit vaccines are excellent vaccine candidates offering effective protection with a better safety profile than live vaccines. In this study, we aim to explore intrinsic adjuvant properties of cationic liposomes to maximize immune activation while minimizing aspecific cytotoxicity. To achieve this, we developed a rational strategy to select liposomal formulation compositions and assessed their physicochemical and immunological properties in vitro models using human monocyte-derived dendritic cells (MDDCs). A broad selection of commercially available cationic compounds was tested to prepare liposomes containing Ag85B-ESAT6-Rv2034 (AER) fusion protein antigen. 1,2-Dioleoyl-sn­glycero-3-ethylphosphocholine (EPC)-based liposomes exhibited the most advantageous activation profile in MDDCs as assessed by cell surface activation markers, cellular uptake, antigen-specific T-cell activation, cytokine production, and cellular viability. The addition of cholesterol to 20 mol% improved the performance of the tested formulations compared to those without it; however, when its concentration was doubled there was no further benefit, resulting in reduced cell viability. This study provides new insights into the role of cationic lipids and cholesterol in liposomal subunit vaccines.

Repurposing diphenylbutylpiperidine-class antipsychotic drugs for host-directed therapy of Mycobacterium tuberculosis and Salmonella enterica infections.

The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide's and Fluspirilene's efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms.

Repurposing diphenylbutylpiperidine-class antipsychotic drugs for host-directed therapy of Mycobacterium tuberculosis and Salmonella enterica infections

The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide’s and Fluspirilene’s efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms.

Differential gene expression of activating Fcγ receptor classifies active tuberculosis regardless of human immunodeficiency virus status or ethnicity.

New diagnostics and vaccines for tuberculosis (TB) are urgently needed, but require an understanding of the requirements for protection from/susceptibility to TB. Previous studies have used unbiased approaches to determine gene signatures in single-site populations. The present study utilized a targeted approach, reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA), to validate these genes in a multisite study. We analysed ex vivo whole blood RNA from a total of 523 participants across four sub-Saharan countries (Ethiopia, Malawi, South Africa, and The Gambia) with differences in TB and human immunodeficiency virus (HIV) status. We found a number of genes that were expressed at significantly lower levels in participants with active disease than in those with latent TB infection (LTBI), with restoration following successful TB treatment. The most consistent classifier of active disease was FCGR1A (high-affinity IgG Fc receptor 1 (CD64)), which was the only marker expressed at significantly higher levels in participants with active TB than in those with LTBI before treatment regardless of HIV status or genetic background. This is the first study to identify a biomarker for TB that is not affected by HIV status or geo-genetic differences. These data provide valuable clues for understanding TB pathogenesis, and also provide a proof-of-concept for the use of RT-MLPA in rapid and inexpensive validation of unbiased gene expression findings.

Identification of biomarkers for tuberculosis disease using a novel dual-color RT-MLPA assay.

Owing to our lack of understanding of the factors that constitute protective immunity during natural infection with Mycobacterium tuberculosis (Mtb), there is an urgent need to identify host biomarkers that predict long-term outcome of infection in the absence of therapy. Moreover, the identification of host biomarkers that predict (in)adequate response to tuberculosis (TB) treatment would similarly be a major step forward. To identify/monitor multi-component host biomarker signatures at the transcriptomic level in large human cohort studies, we have developed and validated a dual-color reverse-transcriptase multiplex ligation-dependent probe amplification (dcRT-MLPA) method, permitting rapid and accurate expression profiling of as many as 60-80 transcripts in a single reaction. dcRT-MLPA is sensitive, highly reproducible, high-throughput, has an extensive dynamic range and is as quantitative as QPCR. We have used dcRT-MLPA to characterize the human immune response to Mtb in several cohort studies in two genetically and geographically diverse populations. A biomarker signature was identified that is strongly associated with active TB disease, and was profoundly distinct from that associated with treated TB disease, latent infection or uninfected controls, demonstrating the discriminating power of our biomarker signature. Identified biomarkers included apoptosis-related genes and T-cell/B-cell markers, suggesting important contributions of adaptive immunity to TB pathogenesis.

A redundant role of the CD3 gamma-immunoreceptor tyrosine-based activation motif in mature T cell function.

At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3gamma, CD3delta, and CD3epsilon) or three (CD3zeta) immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is unresolved: it has been speculated that the different ITAMs function in signal specification, but they may also serve in signal amplification. Because the CD3zeta chains do not contribute unique signaling functions to the TCR, and because the ITAMs of the CD3-gammadeltaepsilon module alone can endow the TCR with normal signaling capacity, it thus becomes important to examine how the CD3gamma-, delta-, and epsilon-ITAMs regulate TCR signaling. We here report on the role of the CD3gamma chain and the CD3gamma-ITAM in peripheral T cell activation and differentiation to effector function. All T cell responses were reduced or abrogated in T cells derived from CD3gamma null-mutant mice, probably because of decreased expression levels of the mature TCR complex lacking CD3gamma. Consistent with this explanation, T cell responses proceed undisturbed in the absence of a functional CD3gamma-ITAM. Loss of integrity of the CD3gamma-ITAM only slightly impaired the regulation of expression of activation markers, suggesting a quantitative contribution of the CD3gamma-ITAM in this process. Nevertheless, the induction of an in vivo T cell response in influenza A virus-infected CD3gamma-ITAM-deficient mice proceeds normally. Therefore, if ITAMs can function in signal specification, it is likely that either the CD3delta and/or the CD3epsilon chains endow the TCR with qualitatively unique signaling functions.

Branching out to gain control: how the pre-TCR is linked to multiple functions.

How is signaling specificity achieved by the pre-TCR during selection of T-cell fate? Like the TCR, this receptor controls many functions, and recent studies define which pathways couple the pre-TCR to the molecular events controlling survival, proliferation, allelic exclusion at the TCRbeta locus, and further differentiation.

Pre-TCR signaling and inactivation of p53 induces crucial cell survival pathways in pre-T cells.

Signaling through the pre-TCR is essential for early T cell development and is severely impaired in mice lacking the CD3 gamma chain of the pre-TCR. We here address the molecular mechanisms underlying this defect. Impaired pre-TCR signaling is shown to be associated with a profound increase in the number of apoptotic CD4- CD8- (DN) thymocytes. Introduction of p53 deficiency into CD3 gamma-deficient mice completely reverses the cell survival defect in CD3 gamma-deficient DN thymocytes and rescues the block in pre-T cell differentiation. In addition, the CD4+ CD8+ (DP) compartment is expanded to its normal size. These findings suggest that the pre-TCR regulates progression through the DNA-damage checkpoint of the DN to DP transition by inactivating p53.

Cell-fate decisions in early T cell development: regulation by cytokine receptors and the pre-TCR.

During lymphocyte development, cell-fate decisions are determined by a myriad of signals produced by the micro- environment of the thymus and the bone marrow. These yet to be fully defined developmental cues regulate stage-specific gene expression, and the extraordinarily well-characterized stages of T and B cell development have provided attractive model systems for studying regulation of cellular differentiation. In particular, studies on the contribution of both antigen receptors and cytokine receptors to lymphoid development have illuminated essential signalling pathways in early T and B cells. Here, we review investigations supporting an obligatory role for the IL-7 receptor pathway in early T cell development. IL-7 is produced by both thymus and bone marrow stromal cells, and its potential contribution to survival, differentiation and proliferation of pro-T cells is discussed. We also address the contribution of the pre-T cell receptor (pre-TCR) to differentiation past the pro-T cell stage, and recent advances in deciphering the composition and function of the pre-TCR complex are discussed. Finally, we suggest future directions in this field that may serve to reveal whether and how signals initiated by the cytokine receptors and pre-TCR may intersect, and to define which down-stream molecular events are regulated by these receptors.

Functional analysis of Ran/TC4 as a protein regulating T-cell costimulation.

Antigen (Ag)-triggered activation of T cells requires engagement of both the T-cell Ag receptor and a costimulatory receptor, for which CD28 can function as a prototypical example. CD80 and CD86 represent ligands for this receptor, and although they are present on professional Ag-presenting cells, these molecules are absent from most tumors. Yet some tumors are still able to costimulate a T-cell response, while others cannot. Therefore, a key question concerns the molecular basis for the costimulation of T cells by those tumor cells not expressing the CD28 ligands CD80 and CD86. Upon screening a cDNA library of such a tumor cell line in a transient COS cell transfection assay for costimulatory activity, we identified Ran/TC4 as a protein whose overexpression results in costimulatory activity. Ran/TC4 is a ubiquitously expressed member of the Ras gene superfamily of small guanosine triphosphate-binding proteins and is involved in nuclear transport; Ran/TC4 cDNA-transfected COS cells specifically costimulate CD8 T cells and not CD4 T cells. Transfection of Ran/TC4 into the costimulation-deficient murine RMA lymphoma cell line introduced costimulatory capacity for CD8 T cells and resulted in markedly elevated levels of nuclear Ran/TC4 protein expression. In addition, in vivo priming of mice with Ran/TC4-transfected RMA cells induced protection against wild-type (wt) RMA tumor cells. Ran/TC4-transfected RMA cells and wt RMA tumor cells exhibit comparable in vivo growth rates in mice lacking T and B cells, and Ran/TC4-mediated tumor rejection thus involves B and/or T cells. This possibility is substantiated by the observation that T cells from normal mice challenged with Ran/TC4-transfected RMA cells can mount a cytotoxic T-cell response not only against the Ran/TC4-transfected tumor cells but also against wt RMA tumor cells. Based on these results, we conclude that gene transfer-mediated elevations in Ran/TC4 can confer costimulatory function for CD8 T cells to tumor cells. This finding suggests a novel application of Ran/TC4 as a protein capable of regulating costimulation in tumor cells.

The CD3gamma chain is essential for development of both the TCRalphabeta and TCRgammadelta lineages.

CD3gamma and CD3delta are the most closely related CD3 components, both of which participate in the TCRalphabeta-CD3 complex expressed on mature T cells. Interestingly, however, CD3delta does not appear to participate functionally in the pre-T-cell receptor (TCR) complex that is expressed on immature T cells: disruption of CD3delta gene expression has no effect on the developmental steps controlled by the pre-TCR. Here we report that in contrast with CD3delta, CD3gamma is an essential component of the pre-TCR. We generated mice selectively lacking expression of CD3gamma, in which expression of CD3delta, CD3epsilon, CD3zeta, pTalpha and TCRbeta remained undisturbed. Thus, all components for composing a pre-TCR are available, with the exception of CD3gamma. Nevertheless, T-cell development is severely inhibited in CD3gamma-deficient mice. The number of cells in the thymus is reduced to <1% of that in normal mice, and the large majority of thymocytes lack CD4 and CD8 and are arrested at the CD44-CD25+ double negative (DN) stage of development. Peripheral lymphoid organs are also practically devoid of T cells, with absolute numbers of peripheral T cells reduced to only 2-5% of those in normal mice. Both TCRalphabeta and TCRgammadelta lineages fail to develop effectively in CD3gamma-deficient mice, although absence of CD3gamma has no effect on gene rearrangements of the TCRbeta, delta and gamma loci. Furthermore, absence of CD3gamma results in a severe reduction in the level of TCR and CD3epsilon expression at the cell surface of thymocytes and peripheral T cells. The defect in the DN to double positive transition in mice lacking CD3gamma can be overcome by anti-CD3epsilon-mediated cross-linking. CD3gamma is thus essential for pre-TCR function.

Induction of TCR gene rearrangements in uncommitted stem cells by a subset of IL-7 producing, MHC class-II-expressing thymic stromal cells.

The embryonic thymic microenvironment provides the necessary elements for T cell lineage commitment, but the precise role of individual stromal cell components remains to be determined. Here we address the question of which stromal cell types are required for initiation of V-DJ rearrangements of the TCR-beta and TCR-delta locus in CD117+CD45+ uncommitted fetal liver progenitors. We show that fetal thymic stroma alone is necessary and sufficient for induction of TCR-beta and TCR-delta rearrangements. Furthermore, the ability to induce this T cell commitment step is confined to a subset of MHC class II-positive epithelial cells. Thymic stroma derived from mice with a targeted deletion in the IL-7 gene, however, lacks this ability. These findings set the stage for a further definition of the nature of the thymic stromal cell support in the regulation of T cell commitment.

Monoclonal antibody EBM11 (anti-CD68) discriminates between dendritic cells and macrophages after short-term culture.

Identification of dendritic cells (DC) is usually done on the basis of their strong MHC class II expression, their typical dendritic morphology and their capacity to induce a strong proliferation of allogeneic T cells. However using these criteria DC can easily be confused with MHC class II positive macrophages (M phi). In addition, the lack of an antibody directed to a specific DC marker greatly hampers the discrimination between DC and M phi. In the present study it is shown that EBM11 (anti-CD68) is a marker specific for both human M phi and DC but in a distinctive way. Human DC locate the EBM11 reactivity in a discrete juxtanuclear spot in contrast to M phi which show EBM11 reactivity throughout the cytoplasm. This greatly improves identification of DC. Light and electron microscopy showed that the CD68 epitope is associated with (phago-)lysosomes. Remarkably the EBM11 spot was only seen in DC after short-term culture, which is an essential step in all classical DC enrichment procedures. Before culture, M phi and DC were indistinguishable. These results show the close relationship between M phi and DC and suggest an important role for the structure of the lysosomal apparatus in these antigen-presenting cells.