Alternating Arenavirus Vector Immunization Generates Robust Polyfunctional Genotype Cross-Reactive Hepatitis B Virus-Specific CD8 T-Cell Responses and High Anti-Hepatitis B Surface Antigen Titers.

Hepatitis B Virus (HBV) is a major driver of infectious disease mortality. Curative therapies are needed and ideally should induce CD8 T cell-mediated clearance of infected hepatocytes plus anti-hepatitis B surface antigen (HBsAg) antibodies (anti-HBs) to neutralize residual virus. We developed a novel therapeutic vaccine using non-replicating arenavirus vectors. Antigens were screened for genotype conservation and magnitude and genotype reactivity of T cell response, then cloned into Pichinde virus (PICV) vectors (recombinant PICV, GS-2829) and lymphocytic choriomeningitis virus (LCMV) vectors (replication-incompetent, GS-6779). Alternating immunizations with GS-2829 and GS-6779 induced high-magnitude HBV T cell responses, and high anti-HBs titers. Dose schedule optimization in macaques achieved strong polyfunctional CD8 T cell responses against core, HBsAg, and polymerase and high titer anti-HBs. In AAV-HBV mice, GS-2829 and GS-6779 were efficacious in animals with low pre-treatment serum HBsAg. Based on these results, GS-2829 and GS-6779 could become a central component of cure regimens.

Constitutive activation of Notch signalling and T cell activation characterize a mouse model of autism.

Gene and protein expression of BTBR T+ Itpr3tf /J (BTBR) mice with autistic-like behaviours were compared with the C57BL/6J strain, which is considered to have normal immunity and behaviour. Notch signalling pathway was constitutively activated in the immune system and liver of BTBR T+ Itpr3tf /J (BTBR) mice. Notch ligand 4 (Dll4), Notch receptors (Notch1 Notch2 and Notch3) and recombination signal binding protein for immunoglobulin κ j region (RBPJ) were increased both at gene and protein levels in BTBR spleens and thymi. Notch downstream transcriptional factors, Tbx21, Gata3, Rorc and FoxP3 were increased in BTBR spleens, Gata3 and FoxP3 were increased in BTBR thymi and BTBR mice have a high blood CD4/CD8 T cell ratio. Reduced nucleotide excision repair ability in BTBR spleens was associated with increased 8-oxoguanine, Ogg1 inhibition, an enhanced level of apoptotic thymocytes and higher expression of GATA-3. Ogg1 inhibition and enhanced GATA-3 expression also were detected in BTBR brain. Notch signal promoted mitochondrial dynamics switching to enhanced fission with an increased number and mass of mitochondria in immune cells of BTBR mice, but not in livers and brains. Constitutive influences on mitochondria exist in this mouse model of autism spectrum disorder; similar outcomes from environmental exposures might occur perinatally in susceptible individuals to affect the development of autism.

Development, phenotypes of immune cells in BTBR T+Itpr3tf/J mice.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition that is characterized by a lack of social interaction, decreased verbal and non-verbal communication skills, and stereotyped repetitive behavior. There is strong evidence that a dysregulated immune response may influence neurodevelopment and thus may have a role in the development of ASD. This study focuses on the characterization of immune cell phenotypes in the BTBR T+Itpr3tf/J (BTBR) mouse strain, a widely used animal model for autism research. Our study demonstrated that BTBR mice have a different immune profile compared to C57BL/6J (B6) mice, which do not display ASD-like characteristics. Thymic cells of BTBR mice have more single positive (SP) CD4+ and CD8+ T cells and fewer double positive (DP) T cells than B6 mice. The development of T cells is increased in BTBR mice with regard to the double negative (DN4) population being much higher in BTBR mice. The spleens and blood of BTBR mice also have more T helper type 1 (Th1), T helper type 2 (Th2) and T regulatory (Treg) cells compared to B6 mice. Aire expression in the thymus and spleen of BTBR mice compared to B6 mice was equivalent and lower, respectively. The mature natural killer (NK) innate immune cell population in blood and spleen is lower in BTBR than B6 mice; NK cell development is blocked prior to the double positive (DN) CD11b+CD27+ stage in BTBR mice. Since BTBR mice have more CD4+ T cells and elevated numbers of Th1 (T-bet+) and Th2 (GATA3+) cells, their low defense against pathogen may be explained by the lower number of NK cells and the significantly lower Th1 to Th2 ratio. The elevated number of plasma cells and autoantibodies of BTBR mice may be due to less presence and function of splenic AIRE.

Transcription factor Bcl11b sustains iNKT1 and iNKT2 cell programs, restricts iNKT17 cell program, and governs iNKT cell survival.

Invariant natural killer T (iNKT) cells are innate-like T cells that recognize glycolipid antigens and play critical roles in regulation of immune responses. Based on expression of the transcription factors (TFs) Tbet, Plzf, and Rorγt, iNKT cells have been classified in effector subsets that emerge in the thymus, namely, iNKT1, iNKT2, and iNKT17. Deficiency in the TF Bcl11b in double-positive (DP) thymocytes has been shown to cause absence of iNKT cells in the thymus and periphery due to defective self glycolipid processing and presentation by DP thymocytes and undefined intrinsic alterations in iNKT precursors. We used a model of cre-mediated postselection deletion of Bcl11b in iNKT cells to determine its intrinsic role in these cells. We found that Bcl11b is expressed equivalently in all three effector iNKT subsets, and its removal caused a reduction in the numbers of iNKT1 and iNKT2 cells, but not in the numbers of iNKT17 cells. Additionally, we show that Bcl11b sustains subset-specific cytokine production by iNKT1 and iNKT2 cells and restricts expression of iNKT17 genes in iNKT1 and iNKT2 subsets, overall restraining the iNKT17 program in iNKT cells. The total numbers of iNKT cells were reduced in the absence of Bcl11b both in the thymus and periphery, associated with the decrease in iNKT1 and iNKT2 cell numbers and decrease in survival, related to changes in survival/apoptosis genes. Thus, these results extend our understanding of the role of Bcl11b in iNKT cells beyond their selection and demonstrate that Bcl11b is a key regulator of iNKT effector subsets, their function, identity, and survival.

Oxidative stress and neuroimmune proteins in a mouse model of autism.

Oxidative stress including decreased antioxidant enzyme activities, elevated lipid peroxidation, and accumulation of advanced glycation end products in the blood from children with autism spectrum disorders (ASD) has been reported. The mechanisms affecting the development of ASD remain unclear; however, toxic environmental exposures leading to oxidative stress have been proposed to play a significant role. The BTBRT+Itpr3tf/J (BTBR) strain provides a model to investigate the markers of oxidation in a mouse strain exhibiting ASD-like behavioral phenotypes. In the present study, we investigated the level of oxidative stress and its effects on immune cell populations, specifically oxidative stress affecting surface thiols (R-SH), intracellular glutathione (iGSH), and expression of brain biomarkers that may contribute to the development of the ASD-like phenotypes that have been observed and reported in BTBR mice. Lower levels of cell surface R-SH were detected on multiple immune cell subpopulations from blood, spleens, and lymph nodes and for sera R-SH levels of BTBR mice compared to C57BL/6 J (B6) mice. The iGSH levels of immune cell populations were also lower in the BTBR mice. Elevated protein expression of GATA3, TGM2, AhR, EPHX2, TSLP, PTEN, IRE1α, GDF15, and metallothionein in BTBR mice is supportive of an increased level of oxidative stress in BTBR mice and may underpin the pro-inflammatory immune state that has been reported in the BTBR strain. Results of a decreased antioxidant system suggest an important oxidative stress role in the development of the BTBR ASD-like phenotype.

Altered meningeal immunity contributing to the autism-like behavior of BTBR T + Itpr3 tf /J mice.

Autism spectrum disorder (ASD) is a complicated neurodevelopmental disorder, which is categorized by deficiency of social contact and communication, and stereotyped forms of performance. Meningeal immunity conditions the immune reflection and immune defense in the meningeal area involving meningeal lymphatic organization, glymphatic structure, immune cells, and cytokines. The development of meningeal immunity dysfunction might be the leading cause for many neural diseases including ASD. The inbred mouse strain BTBRT + Itpr3tf/J (BTBR) shows multiple ASD-like behavioral phenotypes, thus making this strain a widely used animal model for ASD. In our previous study, we reported an altered peripheral immune profile in BTBR mice. Herein, we are investigating immunological and neural interactions associated with the aberrant behavior of BTBR mice. BTBR mice have an increased level of immune cell deposition in the meninges along with a higher level of CD4+ T cells expressing CD25 and of B and myeloid cells expressing more MHCII than C57BL/6 (B6) mice, which have normal behaviors. BTBR mice also have higher levels of autoantibodies to dsDNA, Aquaporin-4, NMDAR1, Pentraxin/SAP and Caspr2 than B6 mice, which may affect neural functions. Interestingly, the T regulatory (Treg) cell population and their function was significantly reduced in the meninges and brain draining lymph nodes, which may explain the increased level of activated B and T cells in the meninges of BTBR mice. A low level of Treg cells, less IL-10 production by Treg, and activated T and B cells in meninges together with higher autoantibody levels might contribute to the development of autism-like behavior through neuroinflammation, which is known to be increased in BTBR mice.

Improvements of autism-like behaviors but limited effects on immune cell metabolism after mitochondrial replacement in BTBR T+Itpr3tf/J mice.

Mitochondria-mediated metabolic impairment and dysfunction are highly related with autism. Herein, the mitochondria-mediated metabolism of BTBR T+Itpr3tf/J (BTBR) mice with autistic-like behaviors was investigated. A new BTBR-mtB6 strain generated by deriving BTBR mice with C57BL/6J (B6) mitochondria was used to determine the role of the mitochondrial genome. The BTBR-mtB6 mice had improved social behaviors, higher levels of glutamate and astrocytes in the brain and less neuroinflammation than the BTBR mice; however, many of the metabolic parameters of BTBR mice such as enhanced fatty acid ß-oxidation and lower glycolysis and glutaminolysis in immune cells compared to B6 mice were not or only partial improved in the BTBR-mtB6 strain. The BTBR and BTBR-mtB6 mice also had equivalent ETC (enhanced electron transport chain) activity of mitochondria, with an increase of reactive oxygen species (ROS) and decreased mitochondrial membrane potential compared to the B6 mice. The results suggest that the mitochondrial replacement with its metabolic alterations affect brain functions more than peripheral immune cell activities.

Gadd34 induces autophagy through the suppression of the mTOR pathway during starvation.

Several types of cellular stress induce expression of growth arrest and DNA damage protein 34 (Gadd34). Autophagy occurs under both basal conditions and conditions of stress, such as starvation. Gadd34 and autophagy are both induced under starvation conditions. In this study we found that starvation induced the expression of Gadd34, reduced mTOR activity, and induced autophagy in wild type mice, but not Gadd34 KO mice. Gadd34 bound to and dephosphorylated pTSC2 at Thr1462. Dephosphorylation of TSC2 during the starvation time period leads to the suppression of mTOR, which is a potent inhibitor of autophagy. We concluded that starvation-induced Gadd34 suppresses mTOR and, thereby, induces autophagy.

Immunity and autoantibodies of a mouse strain with autistic-like behavior.

Female and male mice of the BTBR T + Itpr3 tf /J (BTBR) strain have behaviors that resemble autism spectrum disorder. In comparison to C57BL/6 (B6) mice, BTBR mice have elevated humoral immunity, in that they have naturally high serum IgG levels and generate high levels of IgG antibodies, including autoantibodies to brain antigens. This study focused on the specificities of autoantibodies and the immune cells and their transcription factors that might be responsible for the autoantibodies. BTBR IgG autoantibodies bind to neurons better than microglia and with highest titer to nuclear antigens. Two of the antigens identified were alpha-enolase (ENO1) and dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial (DLST). Surprisingly based on IgG levels, the blood and spleens of BTBR mice have more CD4+ and CD8+ T cells, but fewer B cells than B6 mice. The high levels of autoantibodies in BTBR relates to their splenic T follicular helper (Tfh) cell levels, which likely are responsible for the higher number of plasma cells in BTBR mice than B6 mice. BTBR mice have increased gene expression of interleukin-21 receptor (I l -21 r) and Paired Box 5 (Pax5), which are known to aid B cell differentiation to plasma cells, and an increased Lysine Demethylase 6B (Kdm6b)/DNA Methyltransferase 1 (Dnmt1) ratio, which increases gene expression. Identification of gene expression and immune activities of BTBR mice may aid understanding of mechanisms associated with autism since neuroimmune network interactions have been posited and induction of autoantibodies may drive the neuroinflammation associated with autism.

Recruitment of Gr1(+)CD11b (+)F4/80 (+) population in the bone marrow and spleen by irradiation-induced pulmonary damage.

Radiation-induced lung injury is a kind of sterile inflammation, which may lead to morbidity and mortality. The mechanism by which ionizing radiation activate the immune system is not well understood. In the present study, we have investigated the immunological responses induced by local irradiation-induced damage in mouse lung. The left lungs of C57BL/6 mice were irradiated at a high dose of 100 Gy. The histology of the lungs and spleen showed evidences of alveolar inflammation and congestion at 2 weeks after X-ray treatment. Also, prominent increase in cells expressing the cell surface markers, Gr(+)CD11b(+)F4/80(+) and Ly6C(+) Ly6G(+) were observed 2 weeks after X-ray treatment (100 Gy). Gr1(+)CD11b(+)F4/80(+) cell depletion by clodronate treatment reversed the histological effects and also failed to recruit Gr(+)CD11b(+) cells or F4/80(+) cells caused by irradiation. The origin of recruited Gr1(+)CD11b(+) cells was found to be a mixed resident and recruited phenotype.

Autophagic activity in thymus and liver during aging.

Impaired or deficient autophagy is believed to cause or contribute to aging, as well as several age-related pathologies. Thymic epithelial cells had a high constitutive level of autophagy. The autophagic process may play a supporting role or even a crucial role in the presentation of self-Ags in the thymus to shape the T-cell repertoires. Autophagic activity in the liver is important for the balance of energy and nutrients for basic cell functions. The abundance of autophagic structure in both cortical and medullary thymic epithelial cells and liver with mouse age has not been examined in detail. Here, we demonstrated that the architecture of mouse thymus and liver markedly changed with age. We found that the expression of LC3 detected by immunofluorescence and Western blot analysis was greatly decreased in thymus and liver of 12-month-old mice. The same level of reduction was observed in thymus and liver of 24-month-old mice. Ultrastructure analysis by an electron microscope revealed that the number of autophagic structure/vacuole in total thymic epithelial cells and hepatocytes decrease with age. The age-related decrease of autophagic structure in thymic epithelial cells may cause the reduction of immunocompetent T-cell pool in aged mice. The age-related decrease of autophagy in liver may induce accumulation of cellular materials in liver of aged mice.

Toxic effects of D-galactose on thymus and spleen that resemble aging.

Continuous low-dose injection of d-galactose induces changes in mice that resemble accelerated aging. As such, these mice have been used as models to study mechanisms of aging. Here, we examined whether repeated (daily, for 60 days) subcutaneous injections (at 50 mg D-galactose/kg) into young adult (i.e., 2-month-old) mice induced changes in key immune system organs that were on par with those associated with aging. The results showed that galactose-treated mice develop histologic changes in their thymic cortical and medullary regions; immunohistochemical analysis revealed unorganized distributions of keratin-5 and keratin-8 proteins in the thymus of these hosts. These histological changes in the thymus of D-galactose-treated mice were also observed in the organs of aged (i.e., 24-month-old control mice); however, in this latter group, these changes were accompanied by a strong infiltration of adipose cells. Galactose-treated mice also evinced alterations within their splenic white and red pulp. Further, ultrastructural analyses of the thymus and spleen of the treated mice revealed increases in irregularly shaped lymphocytes bearing visible pyknosis. It was also seen that levels of autophagy within thymic epithelial cells were greatly decreased in the tissues of the galactose-treated mice, an outcome also seen in aged mice. Lastly, the level of memory T-lymphocytes and percentage of IgM-B220-B-lymphocytes in spleens of the galactose-treated mice were both increased (albeit insignificantly so) relative to values among splenocytes of age-matched control; however, these levels were not clearly as elevated as would be expected in "elderly" mice. Taken together, our results strongly suggest that d-galactose treatment can induce structural changes in the thymus and spleen, and some changes in organ-associated cell phenotypes, that are similar to several effects seen with aging. However, the fact that many endpoints do not appear to be truly reflective of what should be seen in immune system organs/cells of "elderly" mice now calls into question the appropriateness of the use of d-galactose (i.e., is it histologically/immunotoxicologically-proper?) to create age-mimicry in mice.