Type I Interferon Signaling Drives Microglial Dysfunction and Senescence in Human iPSC Models of Down Syndrome and Alzheimers Disease

Microglia are critical for brain development and play a central role in Alzheimers disease (AD) etiology. Down syndrome (DS), also known as trisomy 21, is the most common genetic origin of intellectual disability and the most common risk factor for AD. Surprisingly, little information is available on the impact of trisomy of human chromosome 21 (Hsa21) on microglia in DS brain development and AD in DS (DSAD). Using our new induced pluripotent stem cell (iPSC)-based human microglia-containing cerebral organoid and chimeric mouse brain models, here we report that DS microglia exhibit enhanced synaptic pruning function during brain development. Consequently, electrophysiological recordings demonstrate that DS microglial mouse chimeras show impaired synaptic neurotransmission, as compared to control microglial chimeras. Upon being exposed to human brain tissue-derived soluble pathological tau, DS microglia display dystrophic phenotypes in chimeric mouse brains, recapitulating microglial responses seen in human AD and DSAD brain tissues. Further flow cytometry, single-cell RNA-sequencing, and immunohistological analyses of chimeric mouse brains demonstrate that DS microglia undergo cellular senescence and exhibit elevated type I interferon signaling after being challenged by pathological tau. Mechanistically, we find that shRNA-mediated knockdown of Hsa21encoded type I interferon receptor genes, IFNARs, rescues the defective DS microglial phenotypes both during brain development and in response to pathological tau. Our findings provide first in vivo evidence supporting a paradigm shifting theory that human microglia respond to pathological tau by exhibiting accelerated senescence and dystrophic phenotypes. Our results further suggest that targeting IFNARs may improve microglial functions during DS brain development and prevent human microglial senescence in DS individuals with AD.

Longer Poly(U) Stretches in the 3′UTR Are Essential for Replication of the Hepatitis C Virus Genotype 4a Clone in in vitro and in vivo

The 3′ untranslated region (UTR) of the hepatitis C virus (HCV) genome plays a significant role in replication including the poly(U) tract (You and Rice, 2008). Here we established an HCV clone that is infectious in vitro and in vivo, from an Egyptian patient with chronic HCV infection and hepatocellular carcinoma (HCC). First, we inoculated the patient plasma into a humanized chimeric mouse and passaged. We observed HCV genotype 4a propagation in the chimeric mouse sera at 1.7 × 107 copies/mL after 6 weeks. Next, we cloned the entire HCV sequence from the HCV-infected chimeric mouse sera using RT-PCR, and 5′ and 3′ RACE methodologies. We obtained first a shorter clone (HCV-G4 KM short, GenBank: AB795432.1), which contained 9,545 nucleotides with 341 nucleotides of the 5′UTR and 177 nucleotides of the 3′UTR, and this was frequently obtained for unknown reasons. We also obtained a longer clone by dividing the HCV genome into three fragments and the poly (U) sequences. We obtained a longer 3′UTR sequence than that of the HCV-G4 KM short clone, which contained 9,617 nucleotides. This longer clone possessed a 3′-UTR of 249 nucleotides (HCV-G4 KM long, GenBank: AB795432.2), because of a 71-nucleotide longer poly (U) stretch. The HCV-G4-KM long clone, but not the HCV-G4-KM short clone, could establish infection in human hepatoma HuH-7 cells. HCV RNAs carrying a nanoluciferase (NL) reporter were also constructed and higher replication activity was observed with G4-KM long-NL in vitro. Next, both short and long RNAs were intra-hepatically injected into humanized chimeric mice. Viral propagation was only observed for the chimeric mouse injected with the HCV-G4 KM long RNA in the sera after 21 days (1.64 × 106 copies/mL) and continued until 10 weeks post inoculation (wpi; 1.45–4.74 × 107 copies/mL). Moreover, sequencing of the HCV genome in mouse sera at 6 wpi revealed the sequence of the HCV-G4-KM long clone. Thus, the in vitro and in vivo results of this study indicate that the sequence of the HCV-G4-KM long RNA is that of an infectious clone.

CD4+ effector T cells accelerate Alzheimer’s disease in mice

Background Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by pathological deposition of misfolded self-protein amyloid beta (Aβ) which in kind facilitates tau aggregation and neurodegeneration. Neuroinflammation is accepted as a key disease driver caused by innate microglia activation. Recently, adaptive immune alterations have been uncovered that begin early and persist throughout the disease. How these occur and whether they can be harnessed to halt disease progress is unclear. We propose that self-antigens would induct autoreactive effector T cells (Teffs) that drive pro-inflammatory and neurodestructive immunity leading to cognitive impairments. Here, we investigated the role of effector immunity and how it could affect cellular-level disease pathobiology in an AD animal model. Methods In this report, we developed and characterized cloned lines of amyloid beta (Aβ) reactive type 1 T helper (Th1) and type 17 Th (Th17) cells to study their role in AD pathogenesis. The cellular phenotype and antigen-specificity of Aβ-specific Th1 and Th17 clones were confirmed using flow cytometry, immunoblot staining and Aβ T cell epitope loaded haplotype-matched major histocompatibility complex II IAb (MHCII-IAb–KLVFFAEDVGSNKGA) tetramer binding. Aβ-Th1 and Aβ-Th17 clones were adoptively transferred into APP/PS1 double-transgenic mice expressing chimeric mouse/human amyloid precursor protein and mutant human presenilin 1, and the mice were assessed for memory impairments. Finally, blood, spleen, lymph nodes and brain were harvested for immunological, biochemical, and histological analyses. Results The propagated Aβ-Th1 and Aβ-Th17 clones were confirmed stable and long-lived. Treatment of APP/PS1 mice with Aβ reactive Teffs accelerated memory impairment and systemic inflammation, increased amyloid burden, elevated microglia activation, and exacerbated neuroinflammation. Both Th1 and Th17 Aβ-reactive Teffs progressed AD pathology by downregulating anti-inflammatory and immunosuppressive regulatory T cells (Tregs) as recorded in the periphery and within the central nervous system. Conclusions These results underscore an important pathological role for CD4+ Teffs in AD progression. We posit that aberrant disease-associated effector T cell immune responses can be controlled. One solution is by Aβ reactive Tregs. Graphical Abstract

Specificity and Heterogeneity of Trained Immunity in Hematopoietic Stem and Progenitor Cells

When innate immune cells and hematopoietic stem and progenitor cells (HSPCs) are exposed to pathogenic agents, they develop heightened responses to subsequent infection through a process called trained immunity. After exposure to a pathogen, bone marrow derived macrophages (BMDMs) generated from trained HSPCs are capable of enhanced pathogen clearance and cytokine production, while exhibiting persistent metabolic rewiring. Because some models of HSPC trained immunity are dependent on interferon gamma (IFNγ) signaling, and our lab has described extensive changes in HSC self-renewal and differentiation upon IFNγ exposure, we hypothesized that persistent IFNγ signaling induced by chronic infection results in reprogramming of HSPCs, causing improved non-specific immunity. To test our hypothesis, we generated a chimeric mouse model of trained immunity by transplanting control or M. avium-exposed HSPCs into naïve recipient mice. Mice that received M. avium trained HSPCs had decreased bacterial load, less splenomegaly, and fewer granulomas upon subsequent M. avium infection, indicating improved immunity. Furthermore, BMDMs generated from mice trained with a single dose of recombinant IFNγ (rIFNγ) exhibited increased pathogen clearance and metabolic profiles ex vivo. To test if rIFNγ training was sufficient to induce HSPC trained immunity in vivo, we isolated HSPCs from rIFNγ-exposed mice and challenged the recipients 4 months later. These in vivo experiments demonstrated that rIFNγ training was insufficient to induce substantial protection against subsequent M. avium challenge, but still induced BMDM metabolic rewiring four months post HSPC training. Collectively, our studies indicate that there are degrees of training that occur upon IFNγ exposure, likely related to the concentration and duration of the primary stimulus. To assess the specificity of cross protection of HSPC trained immunity, we utilized our chimeric mouse model and tested two different training and infection pathogens: M. avium and influenza. When we challenged M. avium-trained HSPC recipients with influenza, we found that although there was mildly decreased lung histopathology and increased production of IFNγ and TNFα, mice succumbed to infection like untrained controls. When we swapped the order of pathogens, we observed that mice receiving influenza-trained HSPCs produced BMDMs with increased killing capability and systemically higher IL-6 and RANTES levels, but these features were insufficient to significantly reduce bacterial CFU counts upon M. avium challenge. These transplant experiments indicate that trained immunity encoded in HSPCs is pathogen specific. To dissect the mechanism of M. avium-induced trained immunity in HSPCs, we performed RNAseq analysis on M. avium-trained HSPCs post-transplant and cross referenced it with RNAseq and WGBS data on primary M. avium-exposed HSPCs. These studies showed consistent differences in cellular signaling, metabolism, immunity, and antigen processing and presentation in the trained HSPCs, indicating that epigenetic and transcriptional reprogramming induced by M. avium exposure is durable following transplant and secondary challenge. To ascertain whether transcriptional changes are homogeneous throughout the HSC compartment, we completed scRNA-seq on naïve and M. avium-exposed hematopoietic cells. We found that genes upregulated upon M. avium exposure in HSCs, including Batf2 and Cxcl9, were induced in a subset of HSPCs, indicating that there is a heterogeneous response to training within the HSPC pool. Strikingly, the trained immunity signature was maintained in neutrophils and macrophages but lost in mature B cells, indicating specific propagation of genetic signatures induced by training in certain lineages. Finally, we found an emerging population of HSCs with B cell gene signatures upon M. avium exposure. Emergence of this HSPC subpopulation may suggest the development of a cell that acts as a direct intermediate between HSC and B cells following training. Our work shows that trained immunity induced by M. avium and persistent IFNγ signaling is pathogen-specific and heterogeneous among primitive HSPCs. Emergence of specific responder cell populations within the HSPC pool may be responsible for enhanced protection against specific infection stimuli, whereas the presence of non-responders may insure long term health of the HSPC pool. Disclosures No relevant conflicts of interest to declare.

263 ASD141, an innate checkpoint inhibitor, modulates tumor associated myeloid cells through CD11b and enhances current immune checkpoint blockade in preclinical model

BackgroundTumor-associated myeloid cells (TAMCs) are a heterogeneous population of myeloid cells present in the tumor microenvironment (TME). They contribute to immunosuppression and growth of solid tumor. These myeloid cells are highly expressed with CD11b, the alpha-chain of integrin receptor alphaMbeta2 (also known as CD11b/CD18, Mac-1, CR3). It has been suggested that activation of CD11b could facilitate the development of peripheral tolerance by inhibiting T helper 17 differentiation. Antigen-presenting cells (dendritic cells and macrophages) have been shown to enhance T cell proliferation with the treatment of anti-CD11b antibody. Furthermore, CD11b plays a critical role in inflammation by modulating Toll-Like receptor (TLR) responses. High avidity activated form of CD11b leads to a rapid inhibition of TLR signaling by promoting degradation of MyD88 and TRIFs. Therefore, CD11b may serve as an innate checkpoint that function as a negative immune regulator.MethodsIn order to investigate the impact of CD11b in modulating the TME and tumor growth, ASCENDO Biotechnology generated a surrogate chimeric mouse IgG1 antibody, mouse ASD141 (Xi2396), which targets mouse CD11b. These antibodies were then tested in murine MC38 colon cancer.ResultsMouse ASD141 as monotherapy results in statistically significant growth inhibition in murine colon cancer models. Xi2396 remodels the TME by decreasing infiltration of TAMCs, and increased infiltration of dendritic cells (cDCs, NKDCs, and pDCs). Furthermore, Xi2396 also enhanced the antigen presentation ability, which is accompanied by an increased expression of MHCII, CD80 and CD86. These results indicate that the anti-CD11b monoclonal antibody, ASD141, designed to modulate TAMCs of the TME represents a novel approach of cancer immunotherapy.Xi2396 treatment also induced high levels of PD-L1 expression in the TME. Since PD-L1 expression in the TME was associated with response to current immune checkpoint blockades, we sought to determine whether Xi2396 treatment is capable of enhancing anti-tumor response to anti-PD1 therapy. Our results showed that combination of Xi2396 and anti-PD1 synergistically suppressed tumor growth.ConclusionsAltogether, our results provide support for clinical efforts to evaluate ASD141 as an innate immune checkpoint drug, especially in combination with commercial immune checkpoint inhibitors.Ethics ApprovalThis study was approved by National Laboratory Animal Center‘s Institutional Animal Care and Use Committee; approval number NLAC-110-D-006-R2.

B cell depletion in immune-mediated rheumatic diseases and coronavirus disease 2019 (COVID-19)

In patients with immune-mеdiated (autoimmune) rheumatic diseases (IMIRD), there are a number of factors (advanced age, uncontrolled inflammation, initially irreversible damage to internal organs, comorbid pathology, genetic and other factors) that can potentially lead to an increase in “sensitivity” to SARS-CoV -2 (severe acute respiratory syndrome coronavirus-2) and concomitant viral and bacterial infections, an increase in the risk of a severe course of COVID-19 (coronavirus disease 2019), a decrease in the effectiveness of therapy for both IMIRDs and COVID-19. An important area of pharmacotherapy for IMIRDs and other autoimmune diseases is associated with the use of anti-B-cell drugs, primarily rituximab (RTX), which is a chimeric (mouse/human) monoclonal antibody (mAb) to the CD20 antigen of B cells. At present, in Russia, the RTM biosimilar, acellbia (BIOCAD), is widely used, which is not inferior to RTX in terms of efficiency and safety. The problems of anti-B-cell therapy during the COVID-19 pandemic in relation to the risk of infection, severe course and insufficient effectiveness of vaccination against SARSCoV- 2 are considered. According to the recommendations of the Association of Rheumatologists of Russia, a more rigorous assessment of indications for induction and maintenance therapy of RTX therapy and harmonization of the timing of drug administration and vaccination is required.

Mouse CD38-Specific Heavy Chain Antibodies Inhibit CD38 GDPR-Cyclase Activity and Mediate Cytotoxicity Against Tumor Cells

CD38 is the major NAD+-hydrolyzing ecto-enzyme in most mammals. As a type II transmembrane protein, CD38 is also a promising target for the immunotherapy of multiple myeloma (MM). Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Using phage display technology, we isolated 13 mouse CD38-specific nanobodies from immunized llamas and produced these as recombinant chimeric mouse IgG2a heavy chain antibodies (hcAbs). Sequence analysis assigned these hcAbs to five distinct families that bind to three non-overlapping epitopes of CD38. Members of families 4 and 5 inhibit the GDPR-cyclase activity of CD38. Members of families 2, 4 and 5 effectively induce complement-dependent cytotoxicity against CD38-expressing tumor cell lines, while all families effectively induce antibody dependent cellular cytotoxicity. Our hcAbs present unique tools to assess cytotoxicity mechanisms of CD38-specific hcAbs in vivo against tumor cells and potential off-target effects on normal cells expressing CD38 in syngeneic mouse tumor models, i.e. in a fully immunocompetent background.

CD27 expression on Treg cells limits immune responses against tumors

Regulatory T cells (Tregs) suppress immune responses and thus contribute to immune homeostasis. On the downside, Tregs also limit immune responses against tumors promoting the progression of cancer. Among the many mechanisms implied in Treg-mediated suppression, the inhibition of dendritic cells (DCs) has been shown to be central in peripheral tolerance induction as well as in cancers. We have shown previously that the maintenance of peripheral T cell tolerance critically depends on cognate interactions between Tregs and DCs and that the CTL priming by unsuppressed steady state DCs is mediated via CD70. Here, we have investigated whether the CD70/CD27 axis is also involved in Treg-mediated suppression of anti-tumor immunity. Using a mixed bone marrow chimeric mouse model in which we can deplete regulatory T cells in a temporally controlled fashion, we show that Treg-expressed CD27 prevents the breakdown of peripheral tolerance and limits anti-tumor immunity. Furthermore, ablation of Treg expressed CD27 acts synergistically with PD-1 checkpoint inhibition to improve CTL mediated immunity against a solid tumor. Our data thus identify Treg-expressed CD27 as a potential target in cancer immunotherapy. Key messages Treg expressed CD27 maintains steady state DC tolerogenic Treg expressed CD27 limits anti-tumor immunity Ablation of Treg expressed CD27 synergizes with PD-1 blockade to improve CTL mediated tumor control