Enzymatic Activity of HPGD in Treg Cells Suppresses Tconv Cells to Maintain Adipose Tissue Homeostasis and Prevent Metabolic Dysfunction.

Regulatory T cells (Treg cells) are important for preventing autoimmunity and maintaining tissue homeostasis, but whether Treg cells can adopt tissue- or immune-context-specific suppressive mechanisms is unclear. Here, we found that the enzyme hydroxyprostaglandin dehydrogenase (HPGD), which catabolizes prostaglandin E2 (PGE2) into the metabolite 15-keto PGE2, was highly expressed in Treg cells, particularly those in visceral adipose tissue (VAT). Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ)-induced HPGD expression in VAT Treg cells, and consequential Treg-cell-mediated generation of 15-keto PGE2 suppressed conventional T cell activation and proliferation. Conditional deletion of Hpgd in mouse Treg cells resulted in the accumulation of functionally impaired Treg cells specifically in VAT, causing local inflammation and systemic insulin resistance. Consistent with this mechanism, humans with type 2 diabetes showed decreased HPGD expression in Treg cells. These data indicate that HPGD-mediated suppression is a tissue- and context-dependent suppressive mechanism used by Treg cells to maintain adipose tissue homeostasis.

Conditional NKT Cell Depletion in Mice Reveals a Negative Feedback Loop That Regulates CTL Cross-Priming.

NKT cells are unconventional T cells whose biological role is incompletely understood. Similar to TH cells, activated NKT cells can cause dendritic cell (DC) maturation, which is required for effective CTL responses. However, it is unclear whether and how NKT cells affect CTLs downstream of the DC maturation phase. This is partially due to the lack of techniques to conditionally deplete NKT cells in vivo. To overcome this problem, we have developed two approaches for this purpose in mice: the first is based on mixed bone marrow chimeras where Jα18 knockout and depletable CD90 congenic bone marrow is combined, and the second used PLZFCre × iDTR bone marrow chimeras, which target innate-like T cells. Using these tools, we found that NKT cell depletion at 20 h, that is, after initial DC activation, did not render CTLs helpless, as CD40L signaling by non-NKT cells sufficed. Instead, NKT cell depletion even augmented CD8 T cell expansion and cytotoxicity by mechanisms distinct from reduced STAT6 signaling. These findings revealed a negative feedback loop by which NKT cells control CTL cross-priming downstream of DC maturation. The techniques described in this study expand the toolbox to study NKT cells and other unconventional T cell subsets in vivo and uncovered a hidden immunoregulatory mechanism.

CD8+ T-Lymphocyte-Driven Limbic Encephalitis Results in Temporal Lobe Epilepsy.

OBJECTIVE: Limbic encephalitis (LE) comprises a spectrum of inflammatory changes in affected brain structures including the presence of autoantibodies and lymphoid cells. However, the potential of distinct lymphocyte subsets alone to elicit key clinicopathological sequelae of LE potentially inducing temporal lobe epilepsy (TLE) with chronic spontaneous seizures and hippocampal sclerosis (HS) is unresolved. METHODS: Here, we scrutinized pathogenic consequences emerging from CD8+ T cells targeting hippocampal neurons by recombinant adeno-associated virus-mediated expression of the model-autoantigen ovalbumin (OVA) in CA1 neurons of OT-I/RAG1-/- mice (termed "OVA-CD8+ LE model"). RESULTS: Viral-mediated antigen transfer caused dense CD8+ T cell infiltrates confined to the hippocampal formation starting on day 5 after virus transduction. Flow cytometry indicated priming of CD8+ T cells in brain-draining lymph nodes preceding hippocampal invasion. At the acute model stage, the inflammatory process was accompanied by frequent seizure activity and impairment of hippocampal memory skills. Magnetic resonance imaging scans at day 7 of the OVA-CD8+ LE model revealed hippocampal edema and blood-brain barrier disruption that converted into atrophy until day 40. CD8+ T cells specifically targeted OVA-expressing, SIINFEKL-H-2Kb -positive CA1 neurons and caused segmental apoptotic neurodegeneration, astrogliosis, and microglial activation. At the chronic model stage, mice exhibited spontaneous recurrent seizures and persisting memory deficits, and the sclerotic hippocampus was populated with CD8+ T cells escorted by NK cells. INTERPRETATION: These data indicate that a CD8+ T-cell-initiated attack of distinct hippocampal neurons is sufficient to induce LE converting into TLE-HS. Intriguingly, the role of CD8+ T cells exceeds neurotoxic effects and points to their major pathogenic role in TLE following LE. ANN NEUROL 2021;89:666-685.

Splenic Red Pulp Macrophages Cross-Prime Early Effector CTL That Provide Rapid Defense against Viral Infections.

Cross-presentation allows dendritic cells (DCs) to present peptides derived from endocytosed Ags on MHC class I molecules, which is important for activating CTL against viral infections and tumors. Type 1 classical DCs (cDC1), which depend on the transcription factor Batf3, are considered the main cross-presenting cells. In this study, we report that soluble Ags are efficiently cross-presented also by transcription factor SpiC-dependent red pulp macrophages (RPM) of the spleen. In contrast to cDC1, RPM used the mannose receptor for Ag uptake and employed the proteasome- and TAP-dependent cytosolic cross-presentation pathway, previously shown to be used in vitro by bone marrow-derived DCs. In an in vivo vaccination model, both cDC1 and RPM cross-primed CTL efficiently but with distinct kinetics. Within a few days, RPM induced very early effector CTL of a distinct phenotype (Ly6A/E+ Ly6C(+) KLRG1- CD127- CX3CR1- Grz-B+). In an adenoviral infection model, such CTL contained the early viral spread, whereas cDC1 induced short-lived effector CTL that eventually cleared the virus. RPM-induced early effector CTL also contributed to the endogenous antiviral response but not to CTL memory generation. In conclusion, RPM can contribute to antiviral immunity by generating a rapid CTL defense force that contains the virus until cDC1-induced CTL are available to eliminate it. This function can be harnessed for improving vaccination strategies aimed at inducing CTL.

TALEN-mediated genome engineering to generate targeted mice.

Genetic mouse models are critical for biomedical research to understand gene function and pathophysiology. In the last years, the generation of genetic mouse models has been revolutionized by the emergence of transcription activator-like effector nucleases (TALENs). TALENs are programmable, sequence-specific DNA-binding proteins fused to a non-specific endonuclease domain used as powerful tools for site-specific induction of DNA double-strand breaks. These result in disruption of the gene product of the targeted locus by mutations induced during repair by error-prone non-homologous end-joining. Alternatively, these DNA double-strand breaks can be exploited to integrate a user-defined sequence by homologous recombination if an appropriate repair plasmid is provided. In this review, we highlight the major technological improvements for genome editing in murine oocytes which have been achieved using TALENs, discuss current limitations of the technology, suggest strategies to broadly apply TALENs, and describe possible future directions to facilitate gene editing in murine oocytes.

Dual fluorescence reporter mice for Ccl3 transcription, translation, and intercellular communication.

Chemokines guide immune cells during their response against pathogens and tumors. Various techniques exist to determine chemokine production, but none to identify cells that directly sense chemokines in vivo. We have generated CCL3-EASER (ErAse, SEnd, Receive) mice that simultaneously report for Ccl3 transcription and translation, allow identifying Ccl3-sensing cells, and permit inducible deletion of Ccl3-producing cells. We infected these mice with murine cytomegalovirus (mCMV), where Ccl3 and NK cells are critical defense mediators. We found that NK cells transcribed Ccl3 already in homeostasis, but Ccl3 translation required type I interferon signaling in infected organs during early infection. NK cells were both the principal Ccl3 producers and sensors of Ccl3, indicating auto/paracrine communication that amplified NK cell response, and this was essential for the early defense against mCMV. CCL3-EASER mice represent the prototype of a new class of dual fluorescence reporter mice for analyzing cellular communication via chemokines, which may be applied also to other chemokines and disease models.

Genetic engineering as a tool for the generation of mouse models to understand disease phenotypes and gene function.

The usage of mouse models has been vital for biomedical research over the last decades, yet the generation of these models has been extremely difficult and labor-intensive. The identification and generation of nucleases able to introduce site-specific DNA double-strand breaks, particularly the CRIPSR/Cas system, is a major breakthrough for this field as the endogenous DNA repair machinery can be hijacked to specifically introduce genome modifications at these sites. This allows for the time-efficient and cost-efficient generation of mouse models by delivery of designer nucleases together with donor DNA into fertilized oocytes.