Re-defining T-Cell Exhaustion: Subset, Function, and Regulation

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.

Metabolic Reprogramming by the Excessive AMPK Activation Exacerbates Antigen-Specific Memory CD8⁺ T Cell Differentiation after Acute Lymphocytic Choriomeningitis Virus Infection

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.

Extrinsic Acquisition of CD80 by Antigen-Specific CD8⁺ T Cells Regulates Their Recall Immune Responses to Acute Viral Infection

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.

Primera evidencia de infección por el virus de la coriomeningitis linfocítica (arenavirus) en roedores Mus musculus capturados en la zona urbana del municipio de Sincelejo, Sucre, Colombia / First evidence of lymphocytic choriomeningitis virus (Arenavirus) infection in Mus musculus rodents captured in the urban area of the municipality of Sincelejo, Sucre, Colombia

Resumen Introducción. El virus de la coriomeningitis linfocítica es un arenavirus del Viejo Mundo que se hospeda en el ratón casero (Mus musculus), y puede causar infecciones congénitas, hidrocefalia, coriorretinitis y falla orgánica múltiple en pacientes receptores de trasplantes. En Colombia aún no se ha reportado la enfermedad mediante diagnóstico clínico, pero en estudios serológicos se ha detectado la infección por el virus Pichindé en roedores en los departamentos del Cauca y Valle del Cauca, y por el virus Guanarito, en roedores en Córdoba. Objetivo. Detectar el virus de la coriomeningitis linfocítica en M. musculus en el municipio de Sincelejo. Materiales y métodos. Se evaluaron 80 muestras de plasma mediante la prueba ELISA usando antígeno del virus de la coriomeningitis linfocítica. Además, se empleó la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR) anidada en muestras de animales seropositivos y seronegativos para la detección del segmento S. Resultados. Se encontró una seroprevalencia de 10% (8/80) y se detectó el genoma viral en 16 muestras de cerebro; el alineamiento (en la Basic Local Alignment Search Tool, BLAST) y el análisis filogenético (mediante el programa MrBayes, versión 3.2.2) confirmaron que correspondía al virus de la coriomeningitis linfocítica. Conclusión. Los resultados indicaron que la infección por el virus de la coriomeningitis linfocítica en humanos podría ocurrir en el área urbana de Sincelejo, aunque hasta la fecha no se hayan reportado casos.

Abstract Introduction: The lymphocytic choriomeningitis virus is an Old World arenavirus that infects Mus musculus, and can cause congenital hydrocephalus, chorioretinitis and multisystemic failure in transplant human recipients. Although the disease has not been clinically diagnosed in Colombia yet, there have been reports of infection with the Pichindé virus in rodents from Cauca and Valle del Cauca departments, and with the Guanarito virus in rodents from Córdoba department. Objective: To identify the lymphocytic choriomeningitis virus from Mus musculus captured in the municipality of Sincelejo. Materials and methods: We evaluated 80 samples of plasma by ELISA using antigen from lymphocytic choriomeningitis virus. Additionally, a nested RT-PCR was performed to seropositive and seronegative samples for the S-segment. Results: We found a 10% seroprevalence (8/80) and the viral genome was detected in 16 brain samples; the alignment (BLAST) and the phylogenetic analysis (MrBayes, version 3.2.2) confirmed the presence of the lymphocytic choriomeningitis virus. Conclusion: The results indicated that human infection with the lymphocytic choriomeningitis virus in humans could occur in the urban area of Sincelejo, although no cases have been reported so far.

Semi-Functional Quantitative Flow Cytometry Assay for Lymphocytic Choriomeningitis Virus Titration

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.

Enhancing T Cell Immune Responses by B Cell-based Therapeutic Vaccine Against Chronic Virus Infection

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.

Detection of Foreign Antigen-specific CD4+Foxp3+ Regulatory T Cells by MHC Class II Tetramer and Intracellular CD154 Staining

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.

Relationship of an hRAD54 gene polymorphism (2290 C/T) in an Ecuadorian population with chronic myelogenous leukemia

The hRAD54 gene is a key member of the RAD52 epistasis group involved in repair of double-strand breaks (DSB) by homologous recombination (HR). Thus, alterations of the normal function of these genes could generate genetic instability, shifting the normal process of the cell cycle, leading the cells to develop into cancer. In this work we analyzed exon 18 of the hRAD54 gene, which has been previously reported by our group to carry a silent polymorphism, 2290 C/T (Ala730Ala), associated to meningiomas. We performed a PCR-SSCP method to detect the polymorphism in 239 samples including leukemia and normal control population. The results revealed that the 2290 C/T polymorphism has frequencies of 0.1 for the leukemia and 0.1 for the control group. These frequencies show no statistical differences. Additionally, we dissected the leukemia group in chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) to evaluate the polymorphism. The frequencies found in these subgroups were 0.14 for CML and 0.05 for ALL. We found statistically significant differences between CML patients and the control group (p < 0.05) but we did not find significant differences between ALL and the control group (p > 0.05). These results suggest a possible link between the 2290 C/T polymorphism of the hRAD54 gene and CML.

Circulating natural killer and γδ T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus

Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs). Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK) and γδ T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+) subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and γδ T cells, indicating that this is not a pathogenic event. V³9 T cells, representing the majority of circulating γδ T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.

Preparation of H-2Db tetramer and its application in enumerating the CD8+ T cells specific for lymphocytic choriomeningitis virus / 生物工程学报

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.

Comportamiento de cepas argentinas del virus de la Coriomeningitis Linfocitaria en roedores / Behavior of Argentine lymphocytic choriomeningitis virus strains in rodents

La actividad del virus LCM fue informada en Argentina a comienzos de la década del 70 y sólo han sido aisladas cinco cepas a partir del roedor Mus domesticus y dos de humanos. El objetivo de este trabajo consistió en investigar características biológicas de las cepas argentinas de virus LCM para compararlas entre sí y respecto a las cepas históricas WE y Armstrong. En células L 929 se obtuvieron placas bajo agarosa tanto con las cepas humanas como con las cepas de ratón, pero en células Vero sólo se obtuvieron placas con las cepas humanas. No se observó ninguna característica morfométrica de las placas que distinguiera nítidamente a las cepas históricas de las cepas argentinas, ni se observaron diferencias que se relacionen con las especies de origen de las cepas. Las cepas históricas y las cepas argentinas no fueron letales para ratón recién nacido (rrn) generando una infección persistente, según se comprobó al inocular ratones recién nacidos (rrn) por vía intracerebral con cepas de virus LCM y detectarse virus en los cerebros cosechados a diferentes días post inoculación. La única excepción fue la cepa Cba An 13065 que resultó virulenta para rrn ya que con sólo 0.026 UFP se logró 1 DL50. Todas las cepas resultaron letales en ratón adulto (rad), siendo las cepas de ratón más virulentas que las cepas de humanos. Estos resultados permitieron evidenciar el diferente comportamiento en cultivos celulares de las cepas de ratón con respecto a las cepas humanas, e identificar marcadores de virulencia mediante la respuesta a la inoculación por vía intracerebral del rad y del rrn.

The activity of LCM virus was first reported in Argentina at the beginning of the seventies and only five strains have been isolated from rodents Mus domesticus and two from humans. The objective of this paper was to find differential biological characteristics of Argentine strains of LCM virus comparing them in relation to the historical strains WE and Armstrong. Regarding the results obtained in tissue culture, when L 929 cells were used, plaque forming units (PFU) were obtained with human and mouse strains, whilst on Vero cells only human strains developed PFU. Differentials characteristics of historical and Argentine strain's plates were not found, neither differences related to the strain's origin. Neither historical nor Argentine strains were lethal to new-born mice giving a persistent infection, that was demonstrated when we inoculated new-born mouse by intracranial route with different strains of LCM virus and virus was isolated from brains harvested at different days post inoculation. The only exception was Cba An 13065 strain that exhibited virulence in new-born mice, only with 0.026 PFU was obtained 1 DL50. All the strains resulted lethal to adult mice. The mouse strains were more virulent than human strains, being Cba An 13065 the most virulent. These results demonstrate a different behavior in tissue culture between human and mouse strains and allow the identification of virulence markers by intracranial inoculation into new-born or adult mice.

Virus de la coriomeningitis linfocitaria: nuevo teratógeno fetal / Lymphocytic choriomeningitis virus: new fetal teratogen

Lymphocytic choriomeningitis virus (LCMV) is a human zoonosis caused by a rodent-borne arenavirus. It has been associated with both postnatal, as well as in-utero, infection in man. Human infection is acquired after inhalation, ingestion or direct contact with the virus found in the urine, feces and saliva of infected mice, guinea pigs, and hamsters. Congenital LCMV infection should be a diagnostic consideration for infants and children who have chorioretinitis, microcephaly or macrocephaly, hydrocephalus, intracranial calcifications, or nonimmune hydrops fetalis. The diagnosis is made serologically via commercially available immunofluorescent antibody testing. Differentiation of congenital LCMV infection from congenital toxoplasmosis, rubella, cytomegalovirus, herpes simplex virus, enterovirus, human parvovirus B19 and syphilis should be made. Further research is necessary to determine the prevalence of this infection in human and rodent populations and to prospectively delineate the spectrum of congenital infection and its consequences. The medical profession, as well as veterinarians and pet shop owners, must educate the public regarding the hazard that wild, pet and laboratory rodents pose to pregnant women.

Aislamiento del virus de la coriomeningitis linfocitaria de seres humanos / Isolation of lymphocytic choriomeningitis virus from human individuals

The activity of lymphocytic choriomeningitis virus (LCMv) in Argentina has been previously reported on the basis of serological evidence in rodents and humans and the isolation of only one strain of LCMv from a Mus domesticus captured in the province of Cordoba. The aim of this paper was to register patients with serological diagnosis of LCM, to isolate and to identify human strains of LCMv in Argentina. During the last 19 years, 15 cases were diagnosed as LCM by immunoflourescent indirect assay (IFI) and enzyme-linked immunosorbent assay (ELISA) but when neutralizing assay (NT) was incorporated, eight cases were classified as confirmed, three as probable and four as negative. The geographic distribution of the cases included three provinces: Cordoba, Buenos Aires and Santa Fe. Viral isolation was attempted in five patients classified as confirmed and only two resulted positive (P5226 and P8573). They were identified as LCMv by IFI and NT. The coexistence of LCMv with other arenaviruses, such as Junin and Oliveros viruses, in the same area, raises the probability of interactions between them, which could modify the virulence and/or pathogenicity for humans associated to genomic changes. Future studies of antigenic, genomic and virulence variability of different Argentine strains of LCMv, as well as the systematic search for human infection, will contribute to define the importance of this viral agent in our country and to implement control measures.