Proinflammatory cytokines promote TET2-mediated DNA demethylation during CD8 T cell effector differentiation.

To gain insight into the signaling determinants of effector-associated DNA methylation programming among CD8 T cells, we explore the role of interleukin (IL)-12 in the imprinting of IFNg expression during CD8 T cell priming. We observe that anti-CD3/CD28-mediated stimulation of human naive CD8 T cells is not sufficient to induce substantial demethylation of the IFNg promoter. However, anti-CD3/CD28 stimulation in the presence of the inflammatory cytokine, IL-12, results in stable demethylation of the IFNg locus that is commensurate with IFNg expression. IL-12-associated demethylation of the IFNg locus is coupled to cell division through TET2-dependent demethylation in an ex vivo human chimeric antigen receptor T cell model system and an in vivo immunologically competent murine system. Collectively, these data illustrate that IL-12 signaling promotes TET2-mediated effector DNA demethylation programming in CD8 T cells and serve as proof of concept that cytokines can guide induction of epigenetically regulated traits for T cell-based immunotherapies.

Mammalian target of rapamycin complex 1 signalling is essential for germinal centre reaction.

The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that has been shown to be essential for the differentiation and function of various immune cells. Earlier in vitro studies showed that mTOR signalling regulates B-cell biology by supporting their activation and proliferation. However, how mTOR signalling temporally regulates in vivo germinal centre B (GCB) cell development and differentiation into short-lived plasma cells, long-lived plasma cells and memory cells is still not well understood. In this study, we used a combined conditional/inducible knock-out system to investigate the temporal regulation of mTOR complex 1 (mTORC1) in the GCB cell response to acute lymphocytic choriomeningitis virus infection by deleting Raptor, a main component of mTORC1, specifically in B cells in pre- and late GC phase. Early Raptor deficiency strongly inhibited GCB cell proliferation and differentiation and plasma cell differentiation. Nevertheless, late GC Raptor deficiency caused only decreases in the size of memory B cells and long-lived plasma cells through poor maintenance of GCB cells, but it did not change their differentiation. Collectively, our data revealed that mTORC1 signalling supports GCB cell responses at both early and late GC phases during viral infection but does not regulate GCB cell differentiation into memory B cells and plasma cells at the late GC stage.

Acetyl CoA Carboxylase 2 Is Dispensable for CD8+ T Cell Responses.

Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2) regulates fatty acid oxidation (FAO) by inhibiting carnitine palmitoyltransferase 1 (CPT1), a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses.

Acid sphingomyelinase is required for protection of effector memory T cells against glucocorticoid-induced cell death.

The activity of acid sphingomyelinase (aSMase) was previously reported to be involved in glucocorticoid-induced cell death (GICD) of T lymphocytes. This mechanism in turn is believed to contribute to the therapeutic efficacy of glucocorticoids (GCs) in the treatment of inflammatory diseases. In this study, we reassessed the role of aSMase in GICD by using aSMase knockout mice. The absence of aSMase largely abolished the partial protection that effector memory CD4(+) T cells in wild-type mice possess against GICD. Reduced IL-2 secretion by aSMase-deficient CD4(+) T cells suggested that a lack of this important survival factor might be the cause of these cells' enhanced susceptibility to GICD. Indeed, addition of IL-2 restored the protection against GICD, whereas neutralization of IL-2 abrogated the otherwise protective effect seen in wild-type effector memory CD4(+) T cells. The therapeutic implications of the altered sensitivity of aSMase-deficient T cells to GICD were assessed in models of inflammatory disorders; namely, experimental autoimmune encephalomyelitis and acute graft-versus-host disease. Surprisingly, GC treatment was equally efficient in both models in terms of ameliorating the diseases, regardless of the genotype of the T cells. Thus, our data reveal a hitherto unrecognized contribution of aSMase to the sensitivity of effector memory CD4(+) T cells to GICD and call into question the traditionally attributed importance of GICD of T cells to the treatment of inflammatory diseases by GCs.

Tec kinases Itk and Rlk are required for CD8+ T cell responses to virus infection independent of their role in CD4+ T cell help.

Itk and Rlk are members of the Tec kinase family of nonreceptor protein tyrosine kinases that are expressed in T cells, NK cells, and mast cells. These proteins are involved in the regulation of signaling processes downstream of the TCR in CD4(+) T cells, particularly in the phosphorylation of phospholipase C-gamma1 after TCR activation; furthermore, both Itk and Rlk are important in CD4(+) T cell development, differentiation, function, and homeostasis. However, few studies have addressed the roles of these kinases in CD8(+) T cell signaling and function. Using Itk(-/-) and Itk(-/-)Rlk(-/-) mice, we examined the roles of these Tec family kinases in CD8(+) T cells, both in vitro and in vivo. These studies demonstrate that the loss of Itk and Rlk impairs TCR-dependent signaling, causing defects in phospholipase C-gamma1, p38, and ERK activation as well as defects in calcium flux and cytokine production in vitro and expansion and effector cytokine production by CD8(+) T cells in response to viral infection. These defects cannot be rescued by providing virus-specific CD4(+) T cell help, thereby substantiating the important role of Tec kinases in CD8(+) T cell signaling.

Immunoproteasome-deficient mice mount largely normal CD8+ T cell responses to lymphocytic choriomeningitis virus infection and DNA vaccination.

During viral infection, constitutive proteasomes are largely replaced by immunoproteasomes, which display distinct cleavage specificities, resulting in different populations of potential CD8(+) T cell epitope peptides. Immunoproteasomes are believed to be important for the generation of many viral CD8(+) T cell epitopes and have been implicated in shaping the immunodominance hierarchies of CD8(+) T cell responses to influenza virus infection. However, it remains unclear whether these conclusions are generally applicable. In this study we investigated the CD8(+) T cell responses to lymphocytic choriomeningitis virus infection and DNA immunization in wild-type mice and in mice lacking the immunoproteasome subunits LMP2 or LMP7. Although the total number of virus-specific cells was lower in LMP2 knockout mice, consistent with their having lower numbers of naive cells before infection, the kinetics of virus clearance were similar in all three mouse strains, and LMP-deficient mice mounted strong primary and secondary lymphocytic choriomeningitis virus-specific CD8(+) T cell responses. Furthermore, the immunodominance hierarchy of the four investigated epitopes (nuclear protein 396 (NP(396)) > gp33 > gp276 > NP(205)) was well maintained. We observed a slight reduction in the NP(205)-specific response in LMP2-deficient mice, but this had no demonstrable biological consequence. DNA vaccination of LMP2- and LMP7-deficient mice induced CD8(+) T cell responses that were slightly lower than, although not significantly different from, those induced in wild-type mice. Taken together, our results challenge the notion that immunoproteasomes are generally needed for effective antiviral CD8(+) T cell responses and for the shaping of immunodominance hierarchies. We conclude that the immunoproteasome may affect T cell responses to only a limited number of viral epitopes, and we propose that its main biological function may lie elsewhere.

Immunoproteasomes down-regulate presentation of a subdominant T cell epitope from lymphocytic choriomeningitis virus.

The cytotoxic T cell response to pathogens is usually directed against a few immunodominant epitopes, while other potential epitopes are either subdominant or not used at all. In C57BL/6 mice, the acute cytotoxic T cell response against lymphocytic choriomeningitis virus is directed against immunodominant epitopes derived from the glycoprotein (gp33-41) and the nucleoprotein (NP396-404), while the gp276-286 epitope remains subdominant. Despite extensive investigations, the reason for this hierarchy between epitopes is not clear. In this study, we show that the treatment of cells with IFN-gamma enhanced the presentation of gp33-41, whereas presentation of the gp276-286 epitope from the same glycoprotein was markedly reduced. Because proteasomes are crucially involved in epitope generation and because IFN-gamma treatment in vitro and lymphocytic choriomeningitis virus infection in vivo lead to a gradual replacement of constitutive proteasomes by immunoproteasomes, we investigated the role of proteasome composition on epitope hierarchy. Overexpression of the active site subunits of immunoproteasomes LMP2, LMP7, and MECL-1 as well as overexpression of LMP2 alone suppressed the presentation of the gp276-286 epitope. The ability to generate gp276-286-specific CTLs was enhanced in LMP2- and LMP7-deficient mice, and macrophages from these mice showed an elevated presentation of this epitope. In vitro digests demonstrated that fragmentation by immunoproteasomes, but not constitutive proteasomes led to a preferential destruction of the gp276 epitope. Taken together, we show that LMP2 and LMP7 can at least in part determine subdominance and shape the epitope hierarchy of CTL responses in vivo.

Expression of the inducible nitric oxide synthase. Correlation with neuropathology and clinical features in mice with lymphocytic choriomeningitis.

Nitric oxide (NO) synthesized by the cytokine-inducible enzyme, nitric oxide synthase (iNOS), is thought to be a key mediator in the host immune response to infection, in which it may have protective, as well as injurious, actions. We sought evidence for a role of NO in the pathogenesis of lymphocytic choriomeningitis by examining the cerebral expression of the iNOS gene in mice after intracranial inoculation of lymphocytic choriomeningitis virus. In euthymic, but not athymic, mice, elevated expression of iNOS mRNA was observed in the brain by day 5 and increased further to high levels by day 6 postinfection. Of the peripheral organs, only the spleen showed significant increases in iNOS mRNA at day 3 postinfection. In situ hybridization revealed that iNOS RNA expression was restricted to cells within the inflammatory infiltrates and in proximity to areas of lymphocytic choriomeningitis virus infection in the brain. Immunostaining for iNOS protein identified numerous positive cells within the inflammatory infiltrates present in the meninges and choroid plexus. Phenotypic analysis revealed the majority of the iNOS containing cells to be Mac-1 positive. T lymphocytes that belonged to the CD8+ and CD4+ subsets were negative for iNOS expression. The kinetics and distribution of cerebral iNOS expression in lymphocytic choriomeningitis are consistent with a major role for the iNOS/NO pathway in the pathogenesis of this immune-mediated neurologic disease.

Detection of virus-specific RNA-dependent RNA polymerase activity in extracts from cells infected with lymphocytic choriomeningitis virus: in vitro synthesis of full-length viral RNA species.

We have developed an in vitro assay for the lymphocytic choriomeningitis virus (LCMV) RNA-dependent RNA polymerase with ribonucleoprotein complexes extracted from acutely infected tissue culture cells. The RNA products synthesized in vitro corresponded in size to the full-length genomic L and S RNAs and subgenomic NP and GP mRNAs normally produced in vivo during acute LCMV infection. In a temporal analysis spanning the first 72 h of acute infection, the in vitro polymerase activity of ribonucleoprotein complexes was maximal at 16 h and declined significantly at later times. In contrast, the intracellular levels of the viral L protein (the putative polymerase protein) appeared to be maximal at 48 to 72 h postinfection. Our results suggest that the accumulation of L protein correlates with reduced viral replication and transcription at later times in acute infection and may be involved in the transition from acute to persistent LCMV infection.

Expression of cytoplasmic granules with T cell-associated serine proteinase-1 activity in Ly-2+(CD8+) T lymphocytes responding to lymphocytic choriomeningitis virus in vivo.

Monoclonal antibodies (mAb) with specificity for the T cell-associated serine proteinase-1 of the mouse (MTSP-1) were used to study expression and storage of this enzyme in T lymphocytes during lymphocytic choriomeningitis (LCM) virus infection in vivo. Immunohistochemical analysis of splenic tissue at the peak of LCM virus-specific T cell-mediated cytolytic responses, i.e., at day 7 post infection, revealed high numbers of MTSP-1+ T lymphocytes in the interfollicular T cell-dependent area of the spleen. More than 50% of Ly-2+(CD8+) cells but only low numbers of Ly-2-(CD8-) cells, previously enriched by flow cytofluorometry, contained large amounts of cytoplasmic granules which stained both with MTSP-1-specific mAb and the esterase substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester. These data demonstrate that in vivo generated LCM virus-induced cytolytic T lymphocytes develop cytoplasmic storage granules containing MTSP-1 and suggest that the mechanism of granule exocytosis is operative in vivo; possibly MTSP-1 is one effector molecule participating in the Ly-2+(CD8+) T lymphocyte-mediated control of virus infection.

Effects of persistent lymphocytic choriomeningitis virus infection on cholinergic neurons in the mouse.

We have examined the effects of persistent infection with the Armstrong E350 strain of lymphocytic choriomeningitis virus (LCMV) on choline acetyltransferase activity in several regions of Balb/c mouse brain. Despite the presence of high titres of virus in brain for as long as 6 months, and a widespread distribution of virus antigen, no decreases in choline acetyltransferase activity could be demonstrated. The enzyme activity was increased in some regions of brain, showing an effect of the persistent virus infection on a differentiated cell function. Although these data do not suggest a role for LCMV in human neurologic disease, similar studies may allow useful animal models to be conveniently and reproducibly generated.

Regional tyrosine hydroxylase and choline acetyltransferase concentrations in the brains of lymphocytic choriomeningitis-infected mice.

The neurotransmitter biosynthesis enzymes tyrosine hydroxylase and choline acetyltransferase were investigated in selected brain areas of Nya : NYLAR mice infected with lymphocytic choriomeningitis (LCM) virus. Statistically significant alterations in the concentrations of both enzymes occurred in the olfactory, caudate, and neocortical regions at 5 days postinfection. No such alterations occurred in mice given cytoxan (150 mg/kg) 3 days postinfection and examined 5 days postinfection. At 10 days postinfection, however, the cytoxan-treated animals had significantly altered enzyme concentrations in the olfactory region, though not in the caudate or neocortex. This alteration appeared to be transitory, since it was not found in cytoxan-treated animals 60 days postinfection. A possible explanation is that virus production or interference in a brain region cycles over a period of hours or days. Still undetermined is whether these neurochemical changes are a primary effect of the virus or a secondary effect due to the immune response. It is noteworthy that cytoxan caused a marked increase in the enzyme activities studied in most of the brain areas.