A fit of CD4+ T cell immune response to an infection by lymphocytic choriomeningitis virus.

We fit an immune response model to data reporting the CD4+ T cell numbers from the 28 days following the infection of mice with lymphocytic choriomeningitis virus LCMV. We used an ODE model that was previously used to describe qualitatively the behaviour of CD4+ T cells, regulatory T cells (Tregs) and interleukine-2 (IL-2) density. The model considered two clonotypes of T cells in order to fit simultaneously the two time series for the gp61 and NP309 epitopes. We observed the proliferation of T cells and, to a lower extent, Tregs during the immune activation phase following infection and subsequently, during the contraction phase, a smooth transition from faster to slower death rates. The six parameters that were optimized were: the beginning and ending times of the immune response, the growth rate of T cells, their capacity, and the two related with the homeostatic numbers of T cells that respond to each epitope. We showed that the ODE model was able to be calibrated thus providing a quantitative description of the data.

Adenovirus Serotype 5 Vaccination Results in Suboptimal CD4 T Helper 1 Responses in Mice.

Adenovirus serotype 5 (Ad5) is one of the most widely used viral vectors and is known to generate potent T cell responses. While many previous studies have characterized Ad5-induced CD8 T cell responses, there is a relative lack of detailed studies that have analyzed CD4 T cells elicited by Ad5 vaccination. Here, we immunized mice with Ad5 vectors encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) and examined GP-specific CD4 T cell responses elicited by Ad5 vectors and compared them to those induced by an acute LCMV infection. In contrast to LCMV infection, where balanced CD4 T helper 1 (Th1) and T follicular helper (Tfh) responses were induced, Ad5 immunization resulted in a significantly reduced frequency of Th1 cells. CD4 T cells elicited by Ad5 vectors expressed decreased levels of Th1 markers, such as Tim3, SLAM, T-bet, and Ly6C, had smaller amounts of cytotoxic molecules like granzyme B, and produced less interferon gamma than CD4 T cells induced by LCMV infection. This defective CD4 Th1 response appeared to be intrinsic for Ad5 vectors and not a reflection of comparing a nonreplicating vector to a live viral infection, since immunization with a DNA vector expressing LCMV-GP generated efficient CD4 Th1 responses. Analysis at early time points (day 3 or 4) after immunization with Ad5 vectors revealed a defect in the expression of CD25 (interleukin-2 [IL-2] receptor alpha chain) on Ad5-elicited CD4 T cells, and administration of exogenous IL-2 following Ad5 immunization partially restored CD4 Th1 responses. These results suggest that impairment of Th1 commitment after Ad5 immunization could be due to reduced IL-2-mediated signaling.IMPORTANCE During viral infection, generating balanced responses of Th1 and Tfh cells is important to induce effective cell-mediated responses and provide optimal help for antibody responses. In this study, to investigate vaccine-induced CD4 T cell responses, we characterized CD4 T cells after immunization with Ad5 vectors expressing LCMV-GP in mice. Ad5 vectors led to altered effector differentiation of LCMV GP-specific CD4 T cells compared to that during LCMV infection. CD4 T cells following Ad5 immunization exhibited impaired Th1 lineage commitment, generating significantly decreased Th1 responses than those induced by LCMV infection. Our results suggest that suboptimal IL-2 signaling possibly plays a role in reduced Th1 development following Ad5 immunization.

CCR7 expression alters memory CD8 T-cell homeostasis by regulating occupancy in IL-7- and IL-15-dependent niches.

C-C receptor 7 (CCR7) is important to allow T cells and dendritic cells to migrate toward CCL19- and CCL21-producing cells in the T-cell zone of the spleen and lymph nodes. The role of this chemokine receptor in regulating the homeostasis of effector and memory T cells during acute viral infection is poorly defined, however. In this study, we show that CCR7 expression alters memory CD8 T-cell homeostasis following lymphocytic choriomeningitis virus infection. Greater numbers of CCR7-deficient memory T cells were formed and maintained compared with CCR7-sufficient memory T cells, especially in the lung and bone marrow. The CCR7-deficient memory T cells also displayed enhanced rates of homeostatic turnover, which may stem from increased exposure to IL-15 as a consequence of reduced exposure to IL-7, because removal of IL-15, but not of IL-7, normalized the numbers of CCR7-sufficient and CCR7-deficient memory CD8 T cells. This result suggests that IL-15 is the predominant cytokine supporting augmentation of the CCR7(-/-) memory CD8 T-cell pool. Taken together, these data suggest that CCR7 biases memory CD8 T cells toward IL-7-dependent niches over IL-15-dependent niches, which provides insight into the homeostatic regulation of different memory T-cell subsets.

Temporal requirements for B cells in the establishment of CD4 T cell memory.

CD4 T cell memory generation is shaped by a number of factors, including the strength and duration of TCR signaling, as well as the priming environment, all of which can be modified by B cells. Studies using B cell-deficient mice indicate B cells play a critical role in generating effector and memory CD4 T cells; however, when and how B cells are acting to promote these responses has not yet been ascertained. In this study, we use anti-CD20 Ab depletion of B cells at different times following Listeria monocytogenes infection to show that B cells are necessary for the induction of optimal CD4 T cell memory, but not for the transition and maintenance of this population. Importantly, the prerequisite of B cells early postinfection is partially dependent on their expression of MHC class II. B cells are not only required during the priming phase, but also necessary for the initiation of robust secondary responses by memory CD4 T cells. Interestingly, the requirement during the recall response is independent of B cell Ag presentation. Overall, these studies demonstrate the temporally and functionally distinct roles for B cells in regulating CD4 T cell responses.

Negative regulation of type I IFN expression by OASL1 permits chronic viral infection and CD8⁺ T-cell exhaustion.

The type I interferons (IFN-Is) are critical not only in early viral control but also in prolonged T-cell immune responses. However, chronic viral infections such as those of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in humans and lymphocytic choriomeningitis virus (LCMV) in mice overcome this early IFN-I barrier and induce viral persistence and exhaustion of T-cell function. Although various T-cell-intrinsic and -extrinsic factors are known to contribute to induction of chronic conditions, the roles of IFN-I negative regulators in chronic viral infections have been largely unexplored. Herein, we explored whether 2'-5' oligoadenylate synthetase-like 1 (OASL1), a recently defined IFN-I negative regulator, plays a key role in the virus-specific T-cell response and viral defense against chronic LCMV. To this end, we infected Oasl1 knockout and wild-type mice with LCMV CL-13 (a chronic virus) and monitored T-cell responses, serum cytokine levels, and viral titers. LCMV CL-13-infected Oasl1 KO mice displayed a sustained level of serum IFN-I, which was primarily produced by splenic plasmacytoid dendritic cells, during the very early phase of infection (2-3 days post-infection). Oasl1 deficiency also led to the accelerated elimination of viremia and induction of a functional antiviral CD8 T-cell response, which critically depended on IFN-I receptor signaling. Together, these results demonstrate that OASL1-mediated negative regulation of IFN-I production at an early phase of infection permits viral persistence and suppresses T-cell function, suggesting that IFN-I negative regulators, including OASL1, could be exciting new targets for preventing chronic viral infection.

Platelets support a protective immune response to LCMV by preventing splenic necrosis.

Severe arenaviral infections in humans are characterized by clinical findings common to other viral hemorrhagic fevers (VHFs), including thrombocytopenia, leukopenia, skin and internal organ hemorrhages, high viral replication, splenic necrosis, and death. Host responses, rather than direct damage by the arenaviral replication, account for most of the observed pathology, but it is not known what protective roles platelets may have in each of the manifestations. To address this issue in an animal model, we compared nondepleted (100%), partially depleted (15%), and profoundly (< 2.5%) platelet depleted mice infected with the mouse arenavirus lymphocytic choriomeningitis virus (LCMV). Here, we describe that systemic bleedings and death were seen only in those animals receiving the stronger depletion treatment. Furthermore, we showed that the nonhemorrhagic but partially platelet-depleted mice were unable to control the viral replication because of generalized splenic necrosis, affecting innate and adaptive immune cells.These data suggest that, by their supportive roles in hemostasis, platelets may be preventing the severe pathology observed in human arenaviral infections.

Accelerated and improved quantification of lymphocytic choriomeningitis virus (LCMV) titers by flow cytometry.

Lymphocytic choriomeningitis virus (LCMV), a natural murine pathogen, is a member of the Arenavirus family, may cause atypical meningitis in humans, and has been utilized extensively as a model pathogen for the study of virus-induced disease and immune responses. Historically, viral titers have been quantified by a standard plaque assay, but for non-cytopathic viruses including LCMV this requires lengthy incubation, so results cannot be obtained rapidly. Additionally, due to specific technical constraints of the plaque assay including the visual detection format, it has an element of subjectivity along with limited sensitivity. In this study, we describe the development of a FACS-based assay that utilizes detection of LCMV nucleoprotein (NP) expression in infected cells to determine viral titers, and that exhibits several advantages over the standard plaque assay. We show that the LCMV-NP FACS assay is an objective and reproducible detection method that requires smaller sample volumes, exhibits a ∼20-fold increase in sensitivity to and produces results three times faster than the plaque assay. Importantly, when applied to models of acute and chronic LCMV infection, the LCMV-NP FACS assay revealed the presence of infectious virus in samples that were determined to be negative by plaque assay. Therefore, this technique represents an accelerated, enhanced and objective alternative method for detection of infectious LCMV that is amenable to adaptation for other viral infections as well as high throughput diagnostic platforms.

Plasmacytoid dendritic cells control T-cell response to chronic viral infection.

Infections with persistent viruses are a frequent cause of immunosuppression, autoimmune sequelae, and/or neoplastic disease. Plasmacytoid dendritic cells (pDCs) are innate immune cells that produce type I interferon (IFN-I) and other cytokines in response to virus-derived nucleic acids. Persistent viruses often cause depletion or functional impairment of pDCs, but the role of pDCs in the control of these viruses remains unclear. We used conditional targeting of pDC-specific transcription factor E2-2 to generate mice that constitutively lack pDCs in peripheral lymphoid organs and tissues. The profound impact of pDC deficiency on innate antiviral responses was revealed by the failure to control acute infection with the cytopathic mouse hepatitis virus. Furthermore, pDC-deficient animals failed to clear lymphocytic choriomeningitis virus (LCMV) from hematopoietic organs during persistent LCMV infection. This failure was associated with reduced numbers and functionality of LCMV-specific CD4(+) helper T cells and impaired antiviral CD8(+) T-cell responses. Adoptive transfer of LCMV-specific T cells revealed that both CD4(+) and CD8(+) T cells required IFN-I for expansion, but only CD4(+) T cells required the presence of pDCs. In contrast, mice with pDC-specific loss of MHC class II expression supported normal CD4(+) T-cell response to LCMV. These data suggest that pDCs facilitate CD4(+) helper T-cell responses to persistent viruses independently of direct antigen presentation. Thus pDCs provide an essential link between innate and adaptive immunity to chronic viral infection, likely through the secretion of IFN-I and other cytokines.

Androgens suppress antigen-specific T cell responses and IFN-γ production during intracranial LCMV infection.

Intracranial (i.c.) lymphocytic choriomeningitis virus (LCMV) infection of mice results in T cell-driven anorexia and weight loss, which is diminished in males compared to females. We investigated sex-specific effects on antigen-presenting cells (APCs) and T cells after i.c. LCMV infection. Numbers of LCMV-specific T cells, APC activation, and levels of inflammatory cytokines and chemokines in CSF were decreased in males compared to females. Orchidectomy enhanced these immune parameters in males, while dihydrotestosterone treatment of orchidectomized males and intact females decreased some of these parameters. These data suggest that qualitative and quantitative effects of androgens on APCs and T cells may contribute to the well-known, but poorly understood sex differences in immunity and autoimmunity.

Circulating natural killer and gammadelta 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 gammadelta 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 gammadelta T cells, indicating that this is not a pathogenic event. V(3)9 T cells, representing the majority of circulating gammadelta 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.

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.

[Acute myelitis of an unusual cause in a child: the lymphocytic choriomeningitis virus]. / Les myélites aiguës de l'enfant, à propos d'une cause rare: le virus de la chorioméningite lymphocytaire.

UNLABELLED: Acute transverse myelitis is a rare disorder in childhood. It usually occurs as a post-infectious disease, but a precise infectious agent is identified in only 20% of cases. OBSERVATION: The diagnosis of acute transverse myelitis was made in a 5.5-year-old girl who initially presented with left Claude-Bernard-Horner syndrome and meningitis. A few days later, motor and sensory tetraparesia with bladder dysfunction was observed. Magnetic resonance imaging showed a diffuse lesion in the medulla, with a hypersignal in the T2 and a hyposignal in the T1 sequences. Serum analysis showed the presence of a viral infection due to the lymphocytic choriomeningitis (LCM) virus. The outcome was marked by complete recovery of the sensorimotor deficit, but a persistence of the left Claude-Bernard-Horner syndrome. CONCLUSION: In rare cases, the LCM virus is responsible for myelitis. In the present case, the Claude-Bernard-Horner syndrome was secondary to the cervico-medullary lesion. Recent reports in the literature have been discussed, in particular as regards the use of immunomodulatory therapy, which clearly improves patient prognosis.

Animal models using lymphocytic choriomeningitis virus.

This unit includes protocols for inducing systemic infection and persistent infection of mice with lymphocytic choriomeningitis virus (LCMV). Methods used to measure T cell responses to LCMV are then described. A protocol to assess anti-LCMV immunity in vivo is also included. Support protocols for preparing LCMV stocks and measuring LCMV titers using a plaque assay are also included. Finally, a support protocol for detecting anti-LCMV antibodies by ELISA is presented.

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

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 Córdoba. 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: Córdoba, 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.

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. (Au)

CCR2+ and CCR5+ CD8+ T cells increase during viral infection and migrate to sites of infection.

Chemokines and their receptors play a critical role in the selective recruitment of various leukocyte subsets. In this study, we correlated the expression of multiple chemokine and CC chemokine receptor (CCR) genes during the course of intracerebral (i.c.) infection with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV), which are prototypic of a noncytopathic and a cytopathic virus, respectively. Infection of mice with either virus resulted in rapid activation and overlapping cerebral expression of a number of chemokine genes. Infection with VSV i.c. causes a rapidly lethal, T cell-independent encephalitis, and infection resulted in a dramatic early up-regulation of chemokine gene expression. Similar marked up-regulation of chemokine expression was not seen until late after LCMV infection and required the presence of activated T cells. Cerebral CCR gene expression was dominated by CCR1, CCR2 and CCR5. However, despite a stronger initial chemokine signal in VSV-infected mice, only LCMV-induced T cell-dependent inflammation was found to be associated with substantially increased expression of CCR genes. Virus-activated CD8+ T cells were found to express CCR2 and CCR5, whereas activated monocytes/macrophages expressed CCR1 in addition to CCR2 and CCR5. Together, these CCR profiles readily account for the CCR profile prominent during CD8+-dependent CNS inflammation.

High frequency of virus-specific interleukin-2-producing CD4(+) T cells and Th1 dominance during lymphocytic choriomeningitis virus infection.

Analysis of C57BL/6 mice acutely infected with lymphocytic choriomeningitis virus (LCMV) by using intracellular cytokine staining revealed a high frequency (2 to 10%) of CD4(+) T cells secreting the Th1-associated cytokines interleukin-2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha, with no concomitant increase in the frequency of CD4(+) T cells secreting the Th2-associated cytokines IL-4, IL-5, and IL-10 following stimulation with viral peptides. In LCMV-infected C57BL/6 CD8(-/-) mice, more than 20% of the CD4(+) T cells secreted IFN-gamma after viral peptide stimulation, whereas less than 1% of the CD4(+) T cells secreted IL-4 under these same conditions. Mice persistently infected with a high dose of LCMV clone 13 also generated a virtually exclusive Th1 response. Thus, LCMV induces a much more profound virus-specific CD4(+) T-cell response than previously recognized, and it is dramatically skewed to a Th1 phenotype.

Noncytopathic clearance of lymphocytic choriomeningitis virus from the hepatocyte.

We have previously shown that interferon and tumor necrosis factor noncytopathically abolish hepatitis B virus (HBV) replication from the hepatocyte and kidney tubular epithelial cells in vivo. Here we show that a persistent lymphocytic choriomeningitis virus (LCMV) infection is cleared from the hepatocyte noncytopathically when the same cytokines are induced in the liver by antigen-nonspecific stimuli. These results indicate that, like HBV, LCMV is also susceptible to intracellular inactivation by cytokine-induced antiviral mechanisms that are operative in the hepatocyte. In contrast, LCMV is not cleared from intrahepatic nonparenchymal cells or splenocytes, indicating that, unlike the hepatocyte, these cells do not produce the factors required to inactivate LCMV. Antiviral mechanisms like these may have evolved to maintain the functional integrity of vital organs in the face of massive infection.

Humoral immunity due to long-lived plasma cells.

Conventional models suggest that long-term antibody responses are maintained by the continuous differentiation of memory B cells into antibody-secreting plasma cells. This is based on the notion that plasma cells are short-lived and need to be continually replenished by memory B cells. We examined the issue of plasma cell longevity by following the persistence of LCMV-specific antibody and plasma cell numbers after in vivo depletion of memory B cells and by adoptive transfer of virus-specific plasma cells into naive mice. The results show that a substantial fraction of plasma cells can survive and continue to secrete antibody for extended periods of time (>1 year) in the absence of any detectable memory B cells. This study documents the existence of long-lived plasma cells and demonstrates a new mechanism by which humoral immunity is maintained.