Anti-Axl antibody treatment reduces the severity of experimental autoimmune encephalomyelitis.

BACKGROUND: Multiple sclerosis is an immune-mediated disease of the central nervous system (CNS) characterized by inflammation, oligodendrocytes loss, demyelination, and damaged axons. Tyro3, Axl, and MerTK belong to a family of receptor tyrosine kinases that regulate innate immune responses and CNS homeostasis. During experimental autoimmune encephalomyelitis (EAE), the mRNA expression of MerTK, Gas6, and Axl significantly increase, whereas Tyro3 and ProS1 remain unchanged. We have shown that Gas6 is neuroprotective during EAE, and since Gas6 activation of Axl may be necessary for conferring neuroprotection, we sought to determine whether α-Axl or α-MerTK antibodies, shown by others to activate their respective receptors in vivo, could effectively reduce inflammation and neurodegeneration. METHODS: Mice received either α-Axl, α-MerTK, IgG isotype control, or PBS before the onset of EAE symptoms. EAE clinical course, axonal damage, demyelination, cytokine production, and immune cell activation in the CNS were used to determine the severity of EAE. RESULTS: α-Axl antibody treatment significantly decreased the EAE clinical indices of female mice during chronic EAE and of male mice during both acute and chronic phases. The number of days mice were severely paralyzed also significantly decreased with α-Axl treatment. Inflammatory macrophages/microglia and the extent of demyelination significantly decreased in the spinal cords of α-Axl-treated mice during chronic EAE, with no differences in the production of pro-inflammatory cytokines. α-MerTK antibody did not influence EAE induction or progression. CONCLUSION: Our data suggests that the beneficial effect of Gas6/Axl signaling observed in mice administered with Gas6 can be partially preserved by administering an activating α-Axl antibody, but not α-MerTK.

Loss of Gas6 and Axl signaling results in extensive axonal damage, motor deficits, prolonged neuroinflammation, and less remyelination following cuprizone exposure.

The TAM (Tyro3, Axl, and MerTK) family of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and ProS1, are important for innate immune responses and central nervous system (CNS) homeostasis. While only Gas6 directly activates Axl, ProS1 activation of Tyro3/MerTK can indirectly activate Axl through receptor heterodimerization. Therefore, we generated Gas6-/- Axl-/- double knockout (DKO) mice to specifically examine the contribution of this signaling axis while retaining ProS1 signaling through Tyro3 and MerTK. We found that naïve young adult DKO and WT mice have comparable myelination and equal numbers of axons and oligodendrocytes in the corpus callosum. Using the cuprizone model of demyelination/remyelination, transmission electron microscopy revealed extensive axonal swellings containing autophagolysosomes and multivesicular bodies, and fewer myelinated axons in brains of DKO mice at 3-weeks recovery from a 6-week cuprizone diet. Analysis of immunofluorescent staining demonstrated more SMI32+ and APP+ axons and less myelin in the DKO mice. There were no significant differences in the number of GFAP+ astrocytes or Iba1+ microglia/macrophages between the groups of mice. However, at 6-weeks cuprizone and recovery, DKO mice had increased proinflammatory cytokine and altered suppressor of cytokine signaling (SOCS) mRNA expression supporting a role for Gas6-Axl signaling in proinflammatory cytokine suppression. Significant motor deficits in DKO mice relative to WT mice on cuprizone were also observed. These data suggest that Gas6-Axl signaling plays an important role in maintaining axonal integrity and regulating and reducing CNS inflammation that cannot be compensated for by ProS1/Tyro3/MerTK signaling.

Targeted GAS6 delivery to the CNS protects axons from damage during experimental autoimmune encephalomyelitis.

Growth arrest-specific protein 6 (GAS6) is a soluble agonist of the TYRO3, AXL, MERTK (TAM) family of receptor tyrosine kinases identified to have anti-inflammatory, neuroprotective, and promyelinating properties. During experimental autoimmune encephalomyelitis (EAE), wild-type (WT) mice demonstrate a significant induction of Gas6, Axl, and Mertk but not Pros1 or Tyro3 mRNA. We tested the hypothesis that intracerebroventricular delivery of GAS6 directly into the CNS of WT mice during myelin oligodendrocyte glycoprotein (MOG)-induced EAE would improve the clinical course of disease relative to artificial CSF (ACSF)-treated mice. GAS6 did not delay disease onset, but significantly reduced the clinical scores during peak and chronic EAE. Mice receiving GAS6 for 28 d had preserved SMI31(+) neurofilament immunoreactivity, significantly fewer SMI32(+) axonal swellings and spheroids and less demyelination relative to ACSF-treated mice. Alternate-day subcutaneous IFNß injection did not enhance GAS6 treatment effectiveness. Gas6(-/-) mice sensitized with MOG35-55 peptide exhibit higher clinical scores during late peak to early chronic disease, with significantly increased SMI32(+) axonal swellings and Iba1(+) microglia/macrophages, enhanced expression of several proinflammatory mRNA molecules, and decreased expression of early oligodendrocyte maturation markers relative to WT mouse spinal cords with scores for 8 consecutive days. During acute EAE, flow cytometry showed significantly more macrophages but not T-cell infiltrates in Gas6(-/-) spinal cords than WT spinal cords. Our data are consistent with GAS6 being protective during EAE by dampening the inflammatory response, thereby preserving axonal integrity and myelination.

The control of reactive oxygen species production by SHP-1 in oligodendrocytes.

We have previously described reduced myelination and corresponding myelin basic protein (MBP) expression in the central nervous system of Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) deficient motheaten (me/me) mice compared with normal littermate controls. Deficiency in myelin and MBP expression in both brains and spinal cords of motheaten mice correlated with reduced MBP mRNA expression levels in vivo and in purified oligodendrocytes in vitro. Therefore, SHP-1 activity seems to be a critical regulator of oligodendrocyte gene expression and function. Consistent with this role, this study demonstrates that oligodendrocytes of motheaten mice and SHP-1-depleted N20.1 cells produce higher levels of reactive oxygen species (ROS) and exhibit corresponding markers of increased oxidative stress. In agreement with these findings, we demonstrate that increased production of ROS coincides with ROS-induced signaling pathways known to affect myelin gene expression in oligodendrocytes. Antioxidant treatment of SHP-1-deficient oligodendrocytes reversed the pathological changes in these cells, with increased myelin protein gene expression and decreased expression of nuclear factor (erythroid-2)-related factor 2 (Nrf2) responsive gene, heme oxygenase-1 (HO-1). Furthermore, we demonstrate that SHP-1 is expressed in human white matter oligodendrocytes, and there is a subset of multiple sclerosis subjects that demonstrate a deficiency of SHP-1 in normal-appearing white matter. These studies reveal critical pathways controlled by SHP-1 in oligodendrocytes that relate to susceptibility of SHP-1-deficient mice to both developmental defects in myelination and to inflammatory demyelinating diseases.

Kaposi's sarcoma-associated herpesvirus microRNA single-nucleotide polymorphisms identified in clinical samples can affect microRNA processing, level of expression, and silencing activity.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that can produce 25 KSHV mature microRNAs. We previously reported single-nucleotide polymorphisms (SNPs) in KSHV-encoded pre-microRNA and mature microRNA sequences from clinical samples (V. Marshall et al., J. Infect. Dis., 195:645-659, 2007). To determine whether microRNA SNPs affect pre-microRNA processing and, ultimately, mature microRNA expression levels, we performed a detailed comparative analysis of (i) mature microRNA expression levels, (ii) in vitro Drosha/Dicer processing, and (iii) RNA-induced silencing complex-dependent targeting of wild-type (wt) and variant microRNA genes. Expression of pairs of wt and variant pre-microRNAs from retroviral vectors and measurement of KSHV mature microRNA expression by real-time reverse transcription-PCR (RT-PCR) revealed differential expression levels that correlated with the presence of specific sequence polymorphisms. Measurement of KSHV mature microRNA expression in a panel of primary effusion lymphoma cell lines by real-time RT-PCR recapitulated some observed expression differences but suggested a more complex relationship between sequence differences and expression of mature microRNA. Furthermore, in vitro maturation assays demonstrated significant SNP-associated changes in Drosha/DGCR8 and/or Dicer processing. These data demonstrate that SNPs within KSHV-encoded pre-microRNAs are associated with differential microRNA expression levels. Given the multiple reports on the involvement of microRNAs in cancer, the biological significance of these phenotypic and genotypic variants merits further studies in patients with KSHV-associated malignancies.

Sequence analysis of Kaposi sarcoma-associated herpesvirus (KSHV) microRNAs in patients with multicentric Castleman disease and KSHV-associated inflammatory cytokine syndrome.

BACKGROUND: Kaposi sarcoma-associated herpesvirus (KSHV) encodes 12 pre-microRNAs that yield 25 mature microRNAs. We previously reported phylogenetic analysis of the microRNA-coding region of KSHV from Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD) patients. We observed a high level of conservation for most sequences but also a divergent cluster of 5 KSHV sequences, including 2 from MCD patients. METHODS: KSHV microRNA sequences from 23 MCD patients and 7 patients with a newly described KSHV-associated inflammatory cytokine syndrome (KICS) were examined by amplification, cloning, and sequencing of a 646-bp fragment of K12/T0.7 encoding microRNA-K12-10 and microRNA-K12-12 and a 2.8-kbp fragment containing the remaining 10 pre-microRNAs. RESULTS: Phylogenetic analysis showed a distinct variant cluster consisting exclusively of MCD and KICS patients in all trees. Pearson χ(2) analysis revealed that 40 single-nucleotide polymorphisms (SNPs) at various loci were significantly associated with MCD and KICS risk. Cluster analysis of these SNPs generated several combinations of 3 SNPs as putative indicators of MCD and KICS risk. CONCLUSIONS: These findings show that MCD and KICS patients frequently have unusual KSHV microRNA sequences and suggest an association between the observed sequence variation and risk of MCD and KICS.

In vivo killing of primary HIV-infected cells by peripheral-injected early memory-enriched anti-HIV duoCAR T cells.

HIV-specific chimeric antigen receptor-T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4+ T cells and monocytes/macrophages. Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell-like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).

Kaposi sarcoma (KS)-associated herpesvirus microRNA sequence analysis and KS risk in a European AIDS-KS case control study.

BACKGROUND: We recently identified polymorphisms in Kaposi sarcoma-associated herpesvirus (KSHV)-encoded microRNA (miRNA) sequences from clinical subjects. Here, we examine whether any of these may contribute to KS risk in a European AIDS-KS case-control study. METHODS: KSHV load in peripheral blood was determined by real-time quantitative polymerase chain reaction. Samples that had detectable viral loads were used to amplify the 2.8-kb miRNA encoding region plus a 646-bp fragment of the K12/T0.7 gene. Additionally, we characterized an 840-bp fragment of the K1 gene to determine KSHV subtypes. RESULTS: KSHV DNA was detected in peripheral blood mononuclear cells of 49.6% of case patients and 6.8% of controls, and viral loads tended to be higher in case patients. Sequences from the miRNA-encoding regions were conserved overall, but distinct polymorphisms were detected, some of which occurred in primary miRNAs, pre-miRNAs, or mature miRNAs. CONCLUSIONS: Patients with KS were more likely to have detectable viral loads than were controls without disease. Despite high conservation in KSHV miRNA-encoded sequences, polymorphisms were observed, including some that have been reported elsewhere. Some polymorphisms could affect mature miRNA processing and appear to be associated with KS risk.

Akt3-Mediated Protection Against Inflammatory Demyelinating Disease.

Akt is a serine/threonine protein kinase that plays a major role in regulating multiple cellular processes. While the isoforms Akt1 and Akt2 are involved in apoptosis and insulin signaling, respectively, the role for Akt3 remains uncertain. Akt3 is predominantly expressed in the brain, and total deletion of Akt3 in mice results in a reduction in brain size and neurodegeneration following injury. Previously, we found that Akt3-/- mice have a significantly worse clinical course during myelin-oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), an animal model in which autoreactive immune cells enter the CNS, resulting in inflammation, demyelination, and axonal injury. Spinal cords of Akt3-/- mice are severely demyelinated and have increased inflammation compared to WT, suggesting a neuroprotective role for Akt3 during EAE. To specifically address the role of Akt3 in neuroinflammation and maintaining neuronal integrity, we used several mouse strains with different manipulations to Akt3. During EAE, Akt3 Nmf350 mice (with enhanced Akt3 kinase activity) had lower clinical scores, a lag in disease onset, a delay in the influx of inflammatory cells into the CNS, and less axonal damage compared to WT mice. A significant increased efficiency of differentiation toward FOXP3 expressing iTregs was also observed in Akt3 Nmf350 mice relative to WT. Mice with a conditional deletion of Akt3 in CD4+ T-cells had an earlier onset of EAE symptoms, increased inflammation in the spinal cord and brain, and had fewer FOXP3+ cells and FOXP3 mRNA expression. No difference in EAE outcome was observed when Akt3 expression was deleted in neurons (Syn1-CKO). These results indicate that Akt3 signaling in T-cells and not neurons is necessary for maintaining CNS integrity during an inflammatory demyelinating disease.

Multispecific anti-HIV duoCAR-T cells display broad in vitro antiviral activity and potent in vivo elimination of HIV-infected cells in a humanized mouse model.

Adoptive immunotherapy using chimeric antigen receptor-modified T cells (CAR-T) has made substantial contributions to the treatment of certain B cell malignancies. Such treatment modalities could potentially obviate the need for long-term antiretroviral drug therapy in HIV/AIDS. Here, we report the development of HIV-1-based lentiviral vectors that encode CARs targeting multiple highly conserved sites on the HIV-1 envelope glycoprotein using a two-molecule CAR architecture, termed duoCAR. We show that transduction with lentiviral vectors encoding multispecific anti-HIV duoCARs confer primary T cells with the capacity to potently reduce cellular HIV infection by up to 99% in vitro and >97% in vivo. T cells are the targets of HIV infection, but the transduced T cells are protected from genetically diverse HIV-1 strains. The CAR-T cells also potently eliminated PBMCs infected with broadly neutralizing antibody-resistant HIV strains, including VRC01/3BNC117-resistant HIV-1. Furthermore, multispecific anti-HIV duoCAR-T cells demonstrated long-term control of HIV infection in vivo and prevented the loss of CD4+ T cells during HIV infection using a humanized NSG mouse model of intrasplenic HIV infection. These data suggest that multispecific anti-HIV duoCAR-T cells could be an effective approach for the treatment of patients with HIV-1 infection.

Autophagy Is a Tolerance-Avoidance Mechanism that Modulates TCR-Mediated Signaling and Cell Metabolism to Prevent Induction of T Cell Anergy.

In response to activation, CD4+ T cells upregulate autophagy. However, the functional consequences of that upregulation have not been fully elucidated. In this study, we identify autophagy as a tolerance-avoidance mechanism. Our data show that inhibition of autophagy during CD4+ T cell activation induces a long-lasting state of hypo-responsiveness that is accompanied by the expression of an anergic gene signature. Cells unable to induce autophagy after T cell receptor (TCR) engagement show inefficient mitochondrial respiration and decreased turnover of the protein tyrosine phosphatase PTPN1, which translates into defective TCR-mediated signaling. In vivo, inhibition of autophagy during antigen priming induces T cell anergy and decreases the severity of disease in an experimental autoimmune encephalomyelitis mouse model. Interestingly, CD4+ T cells isolated from the synovial fluid of juvenile idiopathic arthritis patients, while resistant to suboptimal stimulation-induced anergy, can be tolerized with autophagy inhibitors. We propose that autophagy constitutes a tolerance-avoidance mechanism, which determines CD4+ T cell fate.