(Mtb) is the causative agent of tuberculosis (TB), which leads to an estimated 1. 5 million deaths worldwide each year. Although the immune correlates of protection against Mtb infection and TB ...disease have not been well-defined, natural killer (NK) cells are increasingly recognized as a key component of the innate immune response to Mtb and as a link between innate and adaptive immunity. In this study, we evaluated NK cell phenotypic and functional profiles in QuantiFERON-TB (QFT)
and QFT
adults in a TB endemic setting in Kisumu, Kenya, and compared their NK cell responses to those of Mtb-naïve healthy adult controls in the U.S. We used flow cytometry to define the phenotypic profile of NK cells and identified distinct CD56
NK cell phenotypes that differentiated the Kenyan and U.S. groups. Additionally, among Kenyan participants, NK cells from QFT
individuals with latent Mtb infection (LTBI) were characterized by significant downregulation of the natural cytotoxicity receptor NKp46 and the inhibitory receptor TIGIT, compared with QFT
individuals. Moreover, the distinct CD56
phenotypic profiles in Kenyan individuals correlated with dampened NK cell responses to tumor cells and diminished activation, degranulation, and cytokine production following stimulation with Mtb antigens, compared with Mtb-naïve U.S. healthy adult controls. Taken together, these data provide evidence that the phenotypic and functional profiles of NK cells are modified in TB endemic settings and will inform future studies aimed at defining NK cell-mediated immune correlates that may be protective against acquisition of Mtb infection and progression to TB disease.
Schistosoma mansoni (SM) is a parasitic helminth that infects over 200 million people and causes severe morbidity. It undergoes a multi-stage life cycle in human hosts and as such stimulates a ...stage-specific immune response. The human T cell response to SM is complex and varies throughout the life cycle of SM. Relative to the wealth of information regarding the immune response to SM eggs, little is known about the immune response to the adult worm. In addition, while a great deal of research has uncovered mechanisms by which co-infection with helminths modulates immunity to other pathogens, there is a paucity of data on the effect of pathogens on immunity to helminths. As such, we sought to characterize the breadth of the T cell response to SM and determine whether co-infection with Mycobacterium tuberculosis (Mtb) modifies SM-specific T cell responses in a cohort of HIV-uninfected adults in Kisumu, Kenya. SM-infected individuals were categorized into three groups by Mtb infection status: active TB (TB), Interferon-γ Release Assay positive (IGRA+), and Interferon-γ Release Assay negative (IGRA-). U.S. adults that were seronegative for SM antibodies served as naïve controls. We utilized flow cytometry to characterize the T cell repertoire to SM egg and worm antigens. We found that T cells had significantly higher proliferation and cytokine production in response to worm antigen than to egg antigen. The T cell response to SM was dominated by γδ T cells that produced TNFα and IFNγ. Furthermore, we found that in individuals infected with Mtb, γδ T cells proliferated less in response to SM worm antigens and had higher IL-4 production compared to naïve controls. Together these data demonstrate that γδ T cells respond robustly to SM worm antigens and that Mtb infection modifies the γδ T cell response to SM.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Antigen-specific CD4 and CD8 T cells are important components of the immune response to
, yet little information is currently known regarding how the breadth, specificity, phenotype, and function of
...-specific T cells correlate with
infection outcome in humans. To facilitate evaluation of human
-specific T cell responses targeting multiple different Ags, we sought to develop a high throughput and reproducible T cell response spectrum assay requiring low blood sample volumes. We describe here the optimization and standardization of a microtiter plate-based, diluted whole blood stimulation assay utilizing overlapping peptide pools corresponding to a functionally diverse panel of 60
Ags. Using IFN-γ production as a readout of Ag specificity, the assay can be conducted using 50 μl of blood per test condition and can be expanded to accommodate additional Ags. We evaluated the intra- and interassay variability, and implemented testing of the assay in diverse cohorts of
-unexposed healthy adults, foreign-born adults with latent
infection residing in the United States, and tuberculosis household contacts with latent
infection in a tuberculosis-endemic setting in Kenya. The
-specific T cell response spectrum assay further enhances the immunological toolkit available for evaluating
-specific T cell responses across different states of
infection, and can be readily implemented in resource-limited settings. Moreover, application of the assay to longitudinal cohorts will facilitate evaluation of treatment- or vaccine-induced changes in the breadth and specificity of Ag-specific T cell responses, as well as identification of
-specific T cell responses associated with
infection outcomes.
HIV infection is a significant risk factor for reactivation of latent
infection (LTBI) and progression to active tuberculosis disease, yet the mechanisms whereby HIV impairs T cell immunity to
have ...not been fully defined. Evaluation of
-specific CD4 T cells is commonly based on IFN-γ production, yet increasing evidence indicates the immune response to
is heterogeneous and encompasses IFN-γ-independent responses. We hypothesized that upregulation of surface activation-induced markers (AIM) would facilitate detection of human
-specific CD4 T cells in a cytokine-independent manner in HIV-infected and HIV-uninfected individuals with LTBI. PBMCs from HIV-infected and HIV-uninfected adults in Kenya were stimulated with CFP-10 and ESAT-6 peptides and evaluated by flow cytometry for upregulation of the activation markers CD25, OX40, CD69, and CD40L. Although
-specific IFN-γ and IL-2 production was dampened in HIV-infected individuals,
-specific CD25
OX40
and CD69
CD40L
CD4 T cells were detectable in the AIM assay in both HIV-uninfected and HIV-infected individuals with LTBI. Importantly, the frequency of
-specific AIM
CD4 T cells was not directly impacted by HIV viral load or CD4 count, thus demonstrating the feasibility of AIM assays for analysis of
-specific CD4 T cells across a spectrum of HIV infection states. These data indicate that AIM assays enable identification of
-specific CD4 T cells in a cytokine-independent manner in HIV-uninfected and HIV-infected individuals with LTBI in a high-tuberculosis burden setting, thus facilitating studies to define novel T cell correlates of protection to
and elucidate mechanisms of HIV-associated dysregulation of antimycobacterial immunity.
Monoclonal antibodies (mAbs) have proven to be effective biological reagents in the form of therapeutic drugs and diagnostics for many pathologies, as well as valuable research tools. Existing ...methods for isolating mAb-producing hybridomas are tedious and time consuming. Herein we describe a novel system in which mAb-secreting hybridoma cells were induced to co-express significant amounts of the membrane form of the secreted immunoglobulin (Ig) on their surfaces and are efficiently recovered by fluorescent activated cell sorting (FACS). Fusion of a novel myeloma parent, SP2ab, expressing transgenic Igα and Igβ of the B-cell receptor complex (BCR) with spleen cells resulted in hybridomas demonstrating order of magnitude increases in BCR surface expression. Surface Ig levels correlated with transgenic Igα expression, and these cells also secreted normal levels of mAb. Hundreds of hybridoma lines producing mAbs specific for a variety of antigens were rapidly isolated as single cell-derived clones after FACS. Significant improvements using the Direct Selection of Hybridomas (DiSH) by FACS include reduced time and labor, improved capability of isolating positive hybridomas, and the ease of manipulating cloned cell lines relative to previously existing approaches that require Limiting Dilution Subcloning (LDS).
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Schistosoma mansoni (SM) is a parasitic helminth that infects over 200 million people and causes severe morbidity. It undergoes a multi-stage life cycle in human hosts and as such stimulates a ...stage-specific immune response. The human T cell response to SM is complex and varies throughout the life cycle of SM. Relative to the wealth of information regarding the immune response to SM eggs, little is known about the immune response to the adult worm. In addition, while a great deal of research has uncovered mechanisms by which co-infection with helminths modulates immunity to other pathogens, there is a paucity of data on the effect of pathogens on immunity to helminths. As such, we sought to characterize the breadth of the T cell response to SM and determine whether co-infection with Mycobacterium tuberculosis (Mtb) modifies SM-specific T cell responses in a cohort of HIV-uninfected adults in Kisumu, Kenya. SM-infected individuals were categorized into three groups by Mtb infection status: active TB (TB), Interferon-gamma Release Assay positive (IGRA+), and Interferon-gamma Release Assay negative (IGRA-). U.S. adults that were seronegative for SM antibodies served as na#239;ve controls. We utilized flow cytometry to characterize the T cell repertoire to SM egg and worm antigens. We found that T cells had significantly higher proliferation and cytokine production in response to worm antigen than to egg antigen. The T cell response to SM was dominated by gammadelta T cells that produced TNFalpha and IFNgamma. Furthermore, we found that in individuals infected with Mtb, gammadelta T cells proliferated less in response to SM worm antigens and had higher IL-4 production compared to na#239;ve controls. Together these data demonstrate that gammadelta T cells respond robustly to SM worm antigens and that Mtb infection modifies the gammadelta T cell response to SM.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Antigen (Ag)-specific CD4 and CD8 T cells are important components of the
immune response to
Mycobacterium tuberculosis
(Mtb), yet little
information is currently known regarding how the breadth, ...specificity, phenotype
and function of Mtb-specific T cells correlate with Mtb infection outcome in
humans. To facilitate evaluation of human Mtb-specific T cell responses
targeting multiple different Ags, we sought to develop a high throughput and
reproducible T cell response spectrum assay (RSA) requiring low blood sample
volumes. We describe here the optimization and standardization of a microtiter
plate-based, diluted whole blood stimulation assay utilizing overlapping peptide
pools corresponding to a functionally diverse panel of 60 Mtb Ags. Using
IFN-γ production as a readout of Ag specificity, the assay can be
conducted using 50µl of blood per test condition and can be expanded to
accommodate additional Ags. We evaluated the intra- and inter-assay variability,
and implemented testing of the assay in diverse cohorts of Mtb-unexposed healthy
adults, foreign-born adults with latent Mtb infection (LTBI) residing in the
U.S., and TB household contacts with LTBI in a TB-endemic setting in Kenya. The
Mtb-specific T cell RSA further enhances the immunological toolkit available for
evaluating Mtb-specific T cell responses across different states of Mtb
infection, and can be readily implemented in resource limited settings.
Moreover, application of the assay to longitudinal cohorts will facilitate
evaluation of treatment- or vaccine-induced changes in the breadth and
specificity of Ag-specific T cell responses, as well as identification of
Mtb-specific T cell responses associated with Mtb infection outcomes.
In this article, we consider the intersection of religious coping and the experience of Hurricanes Katrina and Rita in a lifespan sample of adults living in south Louisiana during the 2005 storms. ...Participants were young, middle-age, older, and oldest-old adults who were interviewed during the post-disaster recovery period. Qualitative analyses confirmed that three dimensions of religion were represented across participants' responses. These dimensions included: (1) faith community, in relation to the significant relief effort and involvement of area churches; (2) religious practices, in the sense of participants' behavioral responses to the storms, such as prayer; and (3) spiritual beliefs, referring to faith as a mechanism underlying individual and family-level adjustment, acceptance, and personal growth in the post-disaster period. Implications for future disaster preparedness are considered.
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BFBNIB, NUK, PILJ, SAZU, UL, UM, UPUK
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