High Diversity and Ancient Common Ancestry of Lymphocytic Choriomeningitis Virus

Lymphocytic choriomeningitis virus (LCMV) is the prototype of the family Arenaviridae. LCMV can be associated with severe disease in humans, and its global distribution reflects the broad dispersion of the primary rodent reservoir, the house mouse (Mus musculus). Recent interest in the natural history of the virus has been stimulated by increasing recognition of LCMV infections during pregnancy, and in clusters of LCMV-associated fatal illness among tissue transplant recipients. Despite its public health importance, little is known regarding the genetic diversity or distribution of virus variants. Genomic analysis of 29 LCMV strains collected from a variety of geographic and temporal sources showed these viruses to be highly diverse. Several distinct lineages exist, but there is little correlation with time or place of isolation. Bayesian analysis estimates the most recent common ancestor to be 1,000–5,000 years old, and this long history is consistent with complex phylogeographic relationships of the extant virus isolates.     

CD8 T cell immunodominance shifts during the early stages of acute LCMV infection independently from functional avidity maturation

Virus-specific T cell responses are often directed to a small subset of possible epitopes and their relative magnitude defines their hierarchy. We determined the size and functional avidity of 4 representative peptide-specific CD8⁺ T cell populations in C57BL/6 mice at different time points after lymphocytic choriomeningitis virus (LCMV) infection. We found that the frequency of different peptide-specific T cell populations in the spleen changed independently over the first 8 days after infection. These changes were not associated with a larger or more rapid increase in functional avidity and yet still resulted in a shift in the final immunodominance hierarchy. Thus, the immunodominance observed at the peak of an antiviral T cell response is not necessarily determined by the initial size or rate of functional avidity maturation of peptide-specific T cell populations.     

The immunoproteasome subunit LMP7 is required in the murine thymus for filling up a hole in the T cell repertoire

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine‐inducible variant of the proteasome which has been implicated in regulating inflammatory responses and antigen presentation. In the thymus, medullary thymic epithelial cells (mTECs) and cortical thymic epithelial cells (cTECs) do express different proteasome subunits exerting chymotrypsin‐like activities suggesting distinct functions in thymic T cell selection. Employing the lymphocytic choriomeningitis virus (LCMV) infection model, we could show that the immunoproteasome subunit LMP7 was absolutely required for the generation of LCMV GP_118‐125‐specific T cells although the class I mediated presentation of GP_118‐125 was not dependent on LMP7. Using bone marrow chimeras and adoptive transfer of LMP7‐deficient CD8⁺ T cells into RAG1‐deficient mice we show that LMP7‐deficient mice lacked GP_118‐125‐specific T cell precursors and that LMP7 was required in radioresistant cells – most likely thymic epithelial cells ‐ to enable their selection. Since LMP7 is strongly expressed in negatively selecting mTECs but barely in positively selecting cTECs our data suggest that LMP7 was required to avoid excessive negative selection of GP_118‐125‐specific T cell precursors. Taken together, this study demonstrates that the immunoproteasome is a crucial factor for filling up holes within the cytotoxic T cell repertoire.     

IL‐2 is required for the generation of viral‐specific CD4+ Th1 tissue‐resident memory cells and B cells are essential for maintenance in the lung

CD4⁺ tissue resident cells are an important first line of defense against viral infections in the lungs and are critical for promoting the localization of lung resident CD8⁺ T cells. However, relatively little is known about the signaling programs required for the development of viral‐specific CD4⁺ tissue resident cells in the lungs. Recently, it was shown that signaling through the high affinity IL‐2 receptor is required for the differentiation of lung‐resident Th2 memory (Trm) cells in a murine model of airway inflammation. We therefore tested if IL‐2 signaling is also required for the development of viral antigen‐specific CD4⁺ Th1 cells in the lung after i.n. infection with lymphocytic choriomeningitis virus. These studies demonstrate that Th1 CD4⁺ T cells also require IL‐2 for lung Trm development. Additionally, they show that B cells potently inhibit early Th1 cell lung residency, but are required for the maintenance of a long‐lived population of CD4⁺ Th1 Trm.     

Spleen-derived macrophages are readily polarized into classically activated (M1) or alternatively activated (M2) states

Bone marrow derived macrophages (BM-MΦ) that differentiate from precursor cells can be polarized into classically activated pro-inflammatory (M1) or alternatively activated (M2) states depending upon the cytokine microenvironment. We questioned whether tissue MΦ, such as spleen-derived MΦ (Sp-MΦ), have the ability to differentiate into M1 or M2 cells. We show in response to activation with IFN-gamma (IFN-γ) and lipopolysaccharide (LPS), that the Sp-MΦ readily acquired an M1 status indicated by up-regulation of iNOS mRNA, nitric oxide (NO) production, and the co-stimulatory molecule CD86. Conversely, Sp-MΦ exposed to IL-4 exhibited increased levels of mannose receptor (CD 206), arginase-1 (Arg)-1 mRNA expression, and significant urea production typical of M2 cells. At this stage of differentiation, the M2 Sp-MΦ were more efficient at phagocytosis of cell-associated antigens than their M1 counterparts. This polarization was not indefinite as the cells could revert back to their original state upon the removal of stimuli and exhibited flexibility to convert from M2 to M1. Remarkably, both M1 and M2 Sp-MΦ induced more CD4 expression on their cells surface after stimulation. We also demonstrate that adherent macrophages cultured for a short term in IL-4 enhances ARG-1 and YM-1 mRNA along with increasing urea producing capacity: traits indicative of an M2 phenotype. Moreover, in response to in vivo virus infection, the adherent macrophages obtained from spleens rapidly express iNOS. These results provide new evidence for the polarization capabilities of Sp-MΦ when exposed to pro-inflammatory or anti-inflammatory cytokines.     

Expansion of regulatory T cells in aged mice following influenza infection

While it has been established that Treg cells can down-modulate an immune response, no study has addressed if the observed increase in Treg cells in aged mice is related to the decreased and delayed specific CD8 T cell responses seen following primary influenza infection. In this study, phenotypic characteristics and function of Treg cells were analyzed in young (4-6 months) and aged (18-22 months) mice prior to and during the course of primary influenza infection. Upon infection, aged, but not young, mice have a significant expansion of Treg cells. In addition, Treg cells of aged mice demonstrate both a higher percentage and higher expression per cell of CD69 both at baseline and during infection compared to young mice. However, Treg cells isolated from young and aged mice comparably suppress CD8 T cells and suppression is dose dependent. These results suggest that the increase in the percentage of Treg cells in aged mice may contribute to the diminished CD8 T cell response to primary influenza infection.     

CD8 T cell defect of TNF-α and IL-2 in DNAM-1 deficient mice delays clearance in vivo of a persistent virus infection

DNAM-1 gene-deficient (−/−) mice take significantly longer to clear an acute and persistent LCMV infection in vivo than DNAM-1 +/+ mice. During acute LCMV priming, at the single cell level, DNAM-1 −/− mice made significantly less cytoplasmic CD8 TNF-α and IL-2 but not IFN-γ than their DNAM-1 +/+ counterparts. Restimulated immune memory CD8 T cells from DNAM-1 −/− and DNAM-1 +/+ mice were equivalent in cytolytic activity against LCMV-infected target cells but DNAM-1 −/− CD8 T cells had significant reductions in TNF-α and IL-2 that were associated on adoptive transfer with the inability to terminate the persistent viral infection.     

Intrinsic defects in CD8 T cells with aging contribute to impaired primary antiviral responses

Aging is associated with altered immune responses, particularly with a diminished CD8 T cell response. Although both intrinsic and extrinsic factors are hypothesized to impact this decreased T cell response, the direct evidence of an intrinsic deficiency in virus-specific CD8 T cells is limited. In this study, a TCR transgenic (Tg) P14 mouse model was utilized to compare the activation and proliferation of the Tg CD8 T cells of young and aged P14 mice upon stimulation with antigen or infection with virus. The proliferation of purified Tg CD8 T cells of aged mice was significantly lower than that of young mice when cultured in vitro with both the LCMV specific peptide and antigen presenting cells from young wild type mice. In addition, expression of the activation markers, CD69, CD25, and CD44, was delayed on Tg T cells of aged mice after stimulation. Importantly, while adoptive transfer of purified Tg CD8 T cells of young or aged mice into young wild type mice resulted in expansion of the Tg CD8 T cells of both ages after LCMV infection, the expansion of the Tg T cells from aged mice was significantly decreased compared with that of the Tg T cells from young mice. However, while the number of IFN-γ secreting Tg CD8 T cells from aged mice was significantly decreased compared to that of young mice, the percentages of Tg CD8 T cells producing IFN-γ were similar in young and aged mice, demonstrating that proliferation, but not function, of the Tg CD8 T cells of aged mice was impaired. Importantly, chronological age alone was not sufficient to predict an altered proliferative response; rather, expression of high levels of CD44 on CD8 T cells of aged mice reflected a decreased proliferative response. These results reveal that alterations intrinsic to CD8 T cells can contribute to the age-associated defects in the primary CD8 T cell response during viral infection.