A High Throughput Whole Blood Assay for Analysis of Multiple Antigen-Specific T Cell Responses in Human Mycobacterium tuberculosis Infection.

Antigen-specific CD4 and CD8 T cells are important components of the immune response to Mycobacterium tuberculosis, yet little information is currently known regarding how the breadth, specificity, phenotype, and function of M. tuberculosis-specific T cells correlate with M. tuberculosis infection outcome in humans. To facilitate evaluation of human M. tuberculosis-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 M. tuberculosis 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 M. tuberculosis-unexposed healthy adults, foreign-born adults with latent M. tuberculosis infection residing in the United States, and tuberculosis household contacts with latent M. tuberculosis infection in a tuberculosis-endemic setting in Kenya. The M. tuberculosis-specific T cell response spectrum assay further enhances the immunological toolkit available for evaluating M. tuberculosis-specific T cell responses across different states of M. tuberculosis 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 M. tuberculosis-specific T cell responses associated with M. tuberculosis infection outcomes.

Active Tuberculosis Is Associated with Depletion of HIV-Specific CD4 and CD8 T Cells in People with HIV.

Infection with Mycobacterium tuberculosis (Mtb) in people with HIV (PWH) is associated with depletion of Mtb-specific CD4 T cell responses, increased risk of progression to active tuberculosis (TB) disease, and increased immune activation. Although higher HIV viral loads have been reported in Mtb/HIV co-infection, the extent to which Mtb infection and TB disease impact the frequency and phenotype of HIV-specific T cell responses has not been well described. We enrolled a cohort of PWH in Kenya across a spectrum of Mtb infection states, including those with no evidence of Mtb infection, latent Mtb infection (LTBI), and active pulmonary TB disease, and evaluated the frequency, immune activation, and cytotoxicity phenotype of HIV-specific CD4 and CD8 T cell responses in peripheral blood by flow cytometry. We found evidence of depletion of HIV-specific CD4 and CD8 T cells in people with TB, but not with LTBI. Expression of the immune activation markers human leukocyte antigen-DR isotype (HLA-DR) and Ki67 and of the cytotoxic molecules granzyme B and perforin were increased in total CD4 and CD8 T cell populations in individuals with TB, although expression of these markers by HIV-specific CD4 and CD8 T cells did not differ by Mtb infection status. These data suggest that TB is associated with overall increased T cell activation and cytotoxicity and with depletion of HIV-specific CD4 and CD8 T cells, which may contribute to further impairment of T cell-mediated immune control of HIV replication in the setting of TB.

mRNA-encoded Cas13 can be used to treat dengue infections in mice.

Dengue is a major global health threat, and there are no approved antiviral agents. Prior research using Cas13 only demonstrated dengue mitigation in vitro. Here we demonstrate that systemic delivery of mRNA-encoded Cas13a and guide RNAs formulated in lipid nanoparticles can be used to treat dengue virus (DENV) 2 and 3 in mice. First, we identified guides against DENV 2 and 3 that demonstrated in vitro efficacy. Next, we confirmed that Cas13 enzymatic activity is necessary for DENV 2 or DENV 3 mitigation in vitro. Last, we show that a single dose of lipid-nanoparticle-formulated mRNA-encoded Cas13a and guide RNA, administered 1 day post-infection, promotes survival of all infected animals and serum viral titre decreases on days 2 and 3 post-infection after lethal challenge in mice. Off-target analysis in mice using RNA sequencing showed no collateral cleavage. Overall, these data demonstrate the potential of mRNA-encoded Cas13 as a pan-DENV drug.

Activation-Induced Marker Expression Identifies Mycobacterium tuberculosis-Specific CD4 T Cells in a Cytokine-Independent Manner in HIV-Infected Individuals with Latent Tuberculosis.

HIV infection is a significant risk factor for reactivation of latent Mycobacterium tuberculosis infection (LTBI) and progression to active tuberculosis disease, yet the mechanisms whereby HIV impairs T cell immunity to M. tuberculosis have not been fully defined. Evaluation of M. tuberculosis-specific CD4 T cells is commonly based on IFN-γ production, yet increasing evidence indicates the immune response to M. tuberculosis is heterogeneous and encompasses IFN-γ-independent responses. We hypothesized that upregulation of surface activation-induced markers (AIM) would facilitate detection of human M. tuberculosis-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 M. tuberculosis-specific IFN-γ and IL-2 production was dampened in HIV-infected individuals, M. tuberculosis-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 M. tuberculosis-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 M. tuberculosis-specific CD4 T cells across a spectrum of HIV infection states. These data indicate that AIM assays enable identification of M. tuberculosis-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 M. tuberculosis and elucidate mechanisms of HIV-associated dysregulation of antimycobacterial immunity.

Adults from Kisumu, Kenya have robust γδ T cell responses to Schistosoma mansoni, which are modulated by tuberculosis.

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.

Distinct Human NK Cell Phenotypes and Functional Responses to Mycobacterium tuberculosis in Adults From TB Endemic and Non-endemic Regions.

Mycobacterium tuberculosis (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 CD56dim 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 CD56dim 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.

Engineered cell surface expression of membrane immunoglobulin as a means to identify monoclonal antibody-secreting hybridomas.

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 Igalpha and Igbeta 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 Igalpha 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).