Lymphocytic choriomeningitis virus/physiology* ; Allosteric Regulation ; Nucleotidyltransferases

Quantitative PCR and plaque assay are powerful virological techniques used to measure the load of defective or infectious virus in mouse and human. However, these methods display limitations such as cross contamination and long run-time. Here, we describe a novel technique termed as semi-functional quantitative flow cytometry (SFQF) for the accurate estimation of the quantity of infectious lymphocytic choriomeningitis virus (LCMV). LCMV titration method using flow cytometry was previously developed but has technical shortcomings, owing to the less optimized parameters such as cell overgrowth, plate scale, and detection threshold. Therefore, we first established optimized conditions for SFQF assay using LCMV nucleoprotein (NP)-specific antibody to evaluate the threshold of the virus detection range in the plaque assay. We subsequently demonstrated that the optimization of the method increased the sensitivity of virus detection. We revealed several new advantages of SFQF assay, which overcomes some of the previously contentious points, and established an upgraded version of the previously reported flow cytometric titration assay. This method extends the detection scale to the level of single cell, allowing extension of its application for in vivo detection of infected cells and their phenotypic analysis. Thus, SFQF assay may serve as an alternative analytical tool for ensuring the reliability of LCMV titration and can be used with other types of viruses using target-specific antibodies.
Animals ; Antibodies ; Flow Cytometry* ; Humans ; Lymphocytic choriomeningitis virus* ; Lymphocytic Choriomeningitis* ; Methods ; Mice ; Nucleoproteins ; Polymerase Chain Reaction

During virus infection, T cells must be adapted to activation and lineage differentiation states via metabolic reprogramming. Whereas effector CD8⁺ T cells preferentially use glycolysis for their rapid proliferation, memory CD8⁺ T cells utilize oxidative phosphorylation for their homeostatic maintenance. Particularly, enhanced AMP-activated protein kinase (AMPK) activity promotes the memory T cell response through different pathways. However, the level of AMPK activation required for optimal memory T cell differentiation remains unclear. A new metformin derivative, IM156, formerly known as HL156A, has been reported to ameliorate various types of fibrosis and inhibit in vitro and in vivo tumors by inducing AMPK activation more potently than metformin. Here, we evaluated the in vivo effects of IM156 on antigen-specific CD8⁺ T cells during their effector and memory differentiation after acute lymphocytic choriomeningitis virus infection. Unexpectedly, our results showed that in vivo treatment of IM156 exacerbated the memory differentiation of virus-specific CD8⁺ T cells, resulting in an increase in short-lived effector cells but decrease in memory precursor effector cells. Thus, IM156 treatment impaired the function of virus-specific memory CD8⁺ T cells, indicating that excessive AMPK activation weakens memory T cell differentiation, thereby suppressing recall immune responses. This study suggests that metabolic reprogramming of antigen-specific CD8⁺ T cells by regulating the AMPK pathway should be carefully performed and managed to improve the efficacy of T cell vaccine.
AMP-Activated Protein Kinases ; Cell Differentiation ; Fibrosis ; Glycolysis ; Immunologic Memory ; In Vitro Techniques ; Lymphocytic choriomeningitis virus ; Lymphocytic Choriomeningitis ; Memory ; Metformin ; Oxidative Phosphorylation ; T-Lymphocytes

The unrestricted population of CD4+Foxp3+ regulatory T (Treg) cells, which have been known to control the expression of autoimmune diseases and protective immunity to inflammatory reactions, has led to greater appreciation of functional plasticity. Detecting and/or isolating Ag-specific CD4+Foxp3+ Tregs at the single cell level are required to study their function and plasticity. In this study, we established and compared both MHC class II tetramer and intracellular CD154 staining, in order to detect CD4+Foxp3+ Treg specific for foreign Ag in acute and chronic infections with lymphocytic choriomeningitis virus (LCMV). Our results revealed that MHC class II tetramer staining showed a lower detection rate of LCMV GP66-77-specific CD4+ T cells because most of MHC class II tetramers were unbound and unstable when combined staining was performed with intracellular cytokines. In contrast, intracellular CD154 staining was revealed to be easier and simple for detecting LCMV GP66-77-specific CD4+ T cells, compared to MHC class II tetramer staining. Subsequently, we employed intracellular CD154 staining to detect LCMV GP66-77-specific CD4+Foxp3+ Tregs using Foxp3GFP knock-in mouse, and found that LCMV GP66-77-specific CD4+Foxp3+ Tregs and polyclonal CD4+Foxp3+ Tregs showed differential expansion in mice infected with LCMV Arms or Cl13 at acute (8 and 13 days pi) and chronic phases (35 days pi). Therefore, our results provide insight into the valuable use of intracellular CD154 staining to detect and characterize foreign Ag-specific CD4+Foxp3+ Treg in various models.
Animals ; Arm ; Autoimmune Diseases ; Cytokines ; Lymphocytic choriomeningitis virus ; Mice ; Plastics ; T-Lymphocytes ; T-Lymphocytes, Regulatory*

Acute viral infection or vaccination generates highly functional memory CD8 T cells following the Ag resolution. In contrast, persistent antigenic stimulation in chronic viral infection and cancer leads to a state of T-cell dysfunction termed T-cell exhaustion. We and other have recently identified a novel subset of exhausted CD8 T cells that act as stem cells for maintaining virus-specific CD8 T cells in a mouse model of chronic lymphocytic choriomeningitis virus infection. This stem cell-like CD8 T-cell subset has been also observed in both mouse and human tumor models. Most importantly, in both chronic viral infection and tumor models, the proliferative burst of Ag-specific CD8 T cells driven by PD-1-directed immunotherapy comes exclusively from this stem cell-like CD8 T-cell subset. Therefore, a better understanding of the mechanisms how CD8 T-cell subsets are regulated during chronic viral infection and cancer is required to improve the current immunotherapies that restore the function of exhausted CD8 T cells. In this review, we discuss the differentiation of virus-specific CD8 T cells during chronic viral infection, the characteristics and function of CD8 T-cell subsets, and the therapeutic intervention of PD-1-directed immunotherapy in cancer.
Animals ; Humans ; Immunotherapy ; Lymphocytic choriomeningitis virus ; Memory ; Mice ; Stem Cells ; T-Lymphocyte Subsets ; T-Lymphocytes ; Vaccination

CD80 is mainly expressed on Ag-presenting cells (APCs) as a costimulatory molecule but is also detected on T cells. However, the origin and physiological role of CD80 on CD8⁺ T cells remain unclear. In the present study, we demonstrated that effector and memory CD8⁺ T cells, but not naïve CD8⁺ T cells, displayed CD80 molecules on their surfaces after acute lymphocytic choriomeningitis virus infection. Using adoptive transfer of CD80-knockout (KO) CD8⁺ T cells into a wild type or CD80-KO recipient, we demonstrated that the effector CD8⁺ T cells displayed CD80 by both intrinsic expression and extrinsic acquisition, while memory CD8⁺ T cells displayed CD80 only by extrinsic acquisition. Interestingly, the extrinsic acquisition of CD80 by CD8⁺ T cells was observed only in the lymphoid organs but not in the periphery, indicating the trogocytosis of CD80 molecules via interaction between CD8⁺ T cells and APCs. We compared the recall immune responses by memory CD8⁺ T cells that either extrinsically acquired CD80 or were deficient in CD80, and found that CD80, presented by memory CD8⁺ T cells, played a role in limiting their expansion and IL-2 production upon exposure to secondary challenge. Our study presents the in vivo dynamics of the extrinsic acquisition of CD80 by Ag-specific CD8⁺ T cells and its role in the regulation of recall immune responses in memory CD8+ T cells.
Adoptive Transfer ; Antigens, CD80 ; Interleukin-2 ; Lymphocytic choriomeningitis virus ; Memory ; T-Lymphocytes

Chronic virus infection leads to the functional impairment of dendritic cells (DCs) as well as T cells, limiting the clinical usefulness of DC-based therapeutic vaccine against chronic virus infection. Meanwhile, B cells have been known to maintain the ability to differentiate plasma cells producing antibodies even during chronic virus infection. Previously, alpha-galactosylceramide (alphaGC) and cognate peptide-loaded B cells were comparable to DCs in priming peptide-specific CD8+ T cells as antigen presenting cells (APCs). Here, we investigated whether B cells activated by alphaGC can improve virus-specific T cell immune responses instead of DCs during chronic virus infection. We found that comparable to B cells isolated from naive mice, chronic B cells isolated from chronically infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13) after alphaGC-loading could activate CD1d-restricted invariant natural killer T (iNKT) cells to produce effector cytokines and upregulate co-stimulatory molecules in both naive and chronically infected mice. Similar to naive B cells, chronic B cells efficiently primed LCMV glycoprotein (GP) 33-41-specific P14 CD8+ T cells in vivo, thereby allowing the proliferation of functional CD8+ T cells. Importantly, when alphaGC and cognate epitope-loaded chronic B cells were transferred into chronically infected mice, the mice showed a significant increase in the population of epitope-specific CD8+ T cells and the accelerated control of viremia. Therefore, our studies demonstrate that reciprocal activation between alphaGC-loaded chronic B cells and iNKT cells can strengthen virus-specific T cell immune responses, providing an effective regimen of autologous B cell-based therapeutic vaccine to treat chronic virus infection.
Animals ; Antibodies ; Antigen-Presenting Cells ; B-Lymphocytes ; Clone Cells ; Cytokines ; Dendritic Cells ; Glycoproteins ; Lymphocytic choriomeningitis virus ; Mice ; Natural Killer T-Cells ; Plasma Cells ; T-Lymphocytes ; Viremia

Major histocompatibility complex (MHC) tetramer technology offers a powerful means to study specific T cell populations of interest. To investigate the immune response of H-2Db-restricted CD8+ T cells in immunotherapy, we prepared the H-2Db tetramer and verified its effectiveness in enumerating the CD8+ T cells specific for the lymphocytic choriomeningitis virus (LCMV). First, the cDNA encoding H-2Db heavy chain was cloned by RT-PCR from the spleen of a C57BL/6 mouse. The expression vector for H-2Db-BSP, i.e. the ectodomain of H-2Db fused to a BirA substrate peptide (BSP), was constructed and overexpressed in E. coli BL21(DE3). Then, the denatured H-2Db-BSP was refolded in the presence of human beta2-microglobulin as well as the GP33-41 peptide (KAVYNFATC, KAV) of LCMV. The biotinylated H-2Db/KAV molecules were purified, then bound to streptavidin-PE and tetramerized. Finally, the prepared H-2Db KAV tetramer reagent was verified by detecting the CD8+ T cells specific for HCMV in KAV peptide vaccinated C57BL/6 mouse, with a mouse receiving subcutaneous injection of only adjuvant as negative control. The results showed that the tetramer positive rates were 0.27%, 0.11%, and 0.24% within the CD8+ T cell populations in the peripheral blood, draining lymph nodes, and spleen of vaccinated mouse, respectively. There was only very low background staining (< or = 0.01%) of those samples from the control mouse. Beside, the best results were achieved in the staining of the peripheral blood sample. In conclusion, the established procedure of preparing H-2Db tetramer will facilitate the study of the immune responses of antigen-specific CD8+ T cells in the experimental immunotherapy on the mice with H-2Db allele background.
Animals ; Antibody Specificity ; CD8-Positive T-Lymphocytes ; immunology ; H-2 Antigens ; genetics ; Histocompatibility Antigens Class I ; genetics ; Immunologic Techniques ; Lymphocytic choriomeningitis virus ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; T-Cell Antigen Receptor Specificity ; immunology