Single-Cell CD4 and CD8 T-Cell Secretome Profiling Reveals Temporal and Niche Differences in Acute Myeloid Leukemia Following Immune Checkpoint Blockade Therapy.

Acute myeloid leukemia (AML) is a heterogeneous malignancy of the blood primarily treated with intensive chemotherapy. The allogeneic T-cell antileukemic activity via donor lymphocyte infusions and stem cell transplantation suggests a potential role for checkpoint blockade therapy in AML. While clinical trials employing these treatments have fallen short of expected results, a deeper exploration into the functional states of T cells in AML could bridge this knowledge gap. In this study, we analyzed the polyfunctional activity of T cells in a cohort of patients with relapsed/refractory (RelRef) AML treated on the clinical trial (ClinicalTrials.gov identifier: NCT02397720) of combination therapy using azacitidine and nivolumab (Aza/Nivo). We utilized the single-cell polyfunctional multiplexed immune assay IsoPlexis to evaluate the CD4 and CD8 T cells in peripheral blood and bone marrow samples collected before and after immunotherapy. This revealed at a pseudobulk level that the CD4 T cells exhibited higher functional activity post-immunotherapy (post-IO), suggesting that CD4-directed therapies may play a role in RelRef AML. Additional single-cell analysis revealed significant differences in baseline polyfunctionality in bone marrows of responders as compared with nonresponders for both CD4 and CD8 T cells. Overall, this study highlights the impact of polyfunctional assessment in understanding CD4 and CD8 dynamics in contexts of therapy in AML. SIGNIFICANCE: We found T-cell polyfunctionality differs between local and systemic microenvironments. Enhanced variability in proteomic profiles of bone marrow CD4 T cells post-IO suggests their pivotal role in AML treatment response. Single-cell analysis identified novel CD4 and CD8 T-cell functional groups linked to immunotherapy response within the bone marrow.

Single-Cell Profiling of CD8+ T Cells in Acute Myeloid Leukemia Reveals a Continuous Spectrum of Differentiation and Clonal Hyperexpansion.

Comprehensive investigation of CD8+ T cells in acute myeloid leukemia (AML) is essential for developing immunotherapeutic strategies beyond immune checkpoint blockade. Herein, we performed single-cell RNA profiling of CD8+ T cells from 3 healthy bone marrow donors and 23 newly diagnosed (NewlyDx) and 8 relapsed/refractory (RelRef) patients with AML. Cells coexpressing canonical exhaustion markers formed a cluster constituting <1% of all CD8+ T cells. We identified two effector CD8+ T-cell subsets characterized by distinct cytokine and metabolic profiles that were differentially enriched in NewlyDx and RelRef patients. We refined a 25-gene CD8-derived signature correlating with therapy resistance, including genes associated with activation, chemoresistance, and terminal differentiation. Pseudotemporal trajectory analysis supported enrichment of a terminally differentiated state in CD8+ T cells with high CD8-derived signature expression at relapse or refractory disease. Higher expression of the 25-gene CD8 AML signature correlated with poorer outcomes in previously untreated patients with AML, suggesting that the bona fide state of CD8+ T cells and their degree of differentiation are clinically relevant. Immune clonotype tracking revealed more phenotypic transitions in CD8 clonotypes in NewlyDx than in RelRef patients. Furthermore, CD8+ T cells from RelRef patients had a higher degree of clonal hyperexpansion associated with terminal differentiation and higher CD8-derived signature expression. Clonotype-derived antigen prediction revealed that most previously unreported clonotypes were patient-specific, suggesting significant heterogeneity in AML immunogenicity. Thus, immunologic reconstitution in AML is likely to be most successful at earlier disease stages when CD8+ T cells are less differentiated and have greater capacity for clonotype transitions.

Single-Cell Profiling of CD8+ T Cells in Acute Myeloid Leukemia Reveals a Continuous Spectrum of Differentiation and Clonal Hyperexpansion.

Comprehensive investigation of CD8+ T cells in acute myeloid leukemia (AML) is essential for developing immunotherapeutic strategies beyond immune checkpoint blockade. Herein, we performed single-cell RNA profiling of CD8+ T cells from 3 healthy bone marrow donors and 23 newly diagnosed (NewlyDx) and 8 relapsed/refractory (RelRef) AML patients. Cells co-expressing canonical exhaustion markers formed a cluster constituting <1% of all CD8+ T cells. We identified two effector CD8+ T cell subsets characterized by distinct cytokine and metabolic profiles that were differentially enriched in NewlyDx and RelRef patients. We refined a 25-gene CD8-derived signature correlating with therapy resistance, including genes associated with activation, chemoresistance, and terminal differentiation. Pseudotemporal trajectory analysis supported enrichment of a terminally differentiated state in CD8+ T cells with high CD8-derived signature expression at relapse or refractory disease. Higher expression of the 25-gene CD8 AML signature correlated with poorer outcomes in previously untreated AML patients, suggesting that the bona fide state of CD8+ T cells and their degree of differentiation are clinically relevant. Immune clonotype tracking revealed more phenotypic transitions in CD8 clonotypes in NewlyDx than in RelRef patients. Furthermore, CD8+ T cells from RelRef patients had a higher degree of clonal hyperexpansion associated with terminal differentiation and higher CD8-derived signature expression. Clonotype-derived antigen prediction revealed that most previously unreported clonotypes were patient-specific, suggesting significant heterogeneity in AML immunogenicity. Thus, immunologic reconstitution in AML is likely to be most successful at earlier disease stages when CD8+ T cells are less differentiated and have greater capacity for clonotype transitions.

Granulins rescue inflammation, lysosome dysfunction, and neuropathology in a mouse model of progranulin deficiency.

Progranulin (PGRN) deficiency is linked to neurodegenerative diseases including frontotemporal dementia, Alzheimer's disease, Parkinson's disease, and neuronal ceroid lipofuscinosis. Proper PGRN levels are critical to maintain brain health and neuronal survival, however the function of PGRN is not well understood. PGRN is composed of 7.5 tandem repeat domains, called granulins, and is proteolytically processed into individual granulins inside the lysosome. The neuroprotective effects of full-length PGRN are well-documented, but the role of granulins is still unclear. Here we report, for the first time, that expression of single granulins is sufficient to rescue the full spectrum of disease pathology in mice with complete PGRN deficiency (Grn-/-). Specifically, rAAV delivery of either human granulin-2 or granulin-4 to Grn-/- mouse brain ameliorates lysosome dysfunction, lipid dysregulation, microgliosis, and lipofuscinosis similar to full-length PGRN. These findings support the idea that individual granulins are the functional units of PGRN, likely mediate neuroprotection within the lysosome, and highlight their importance for developing therapeutics to treat FTD-GRN and other neurodegenerative diseases.

Allele-specific cis-regulatory methylation of the gene for vasoactive intestinal peptide in white-throated sparrows.

White-throated sparrows (Zonotrichia albicollis) offer a unique opportunity to connect genotype with behavioral phenotype. In this species, a rearrangement of the second chromosome is linked with territorial aggression; birds with a copy of this "supergene" rearrangement are more aggressive than those without it. The supergene has captured the gene VIP, which encodes vasoactive intestinal peptide, a neuromodulator that drives aggression in other songbirds. In white-throated sparrows, VIP expression is higher in the anterior hypothalamus of birds with the supergene than those without it, and expression of VIP in this region predicts the level of territorial aggression regardless of genotype. Here, we aimed to identify epigenetic mechanisms that could contribute to differential expression of VIP both in breeding adults, which exhibit morph differences in territorial aggression, and in nestlings, before territorial behavior develops. We extracted and bisulfite-converted DNA from samples of the hypothalamus in wild-caught adults and nestlings and used high-throughput sequencing to measure DNA methylation of a region upstream of the VIP start site. We found that the allele inside the supergene was less methylated than the alternative allele in both adults and nestlings. The differential methylation was attributed primarily to CpG sites that were shared between the alleles, not to polymorphic sites, which suggests that epigenetic regulation is occurring independently of the genetic differentiation within the supergene. This work represents an initial step toward understanding how epigenetic differentiation inside chromosomal inversions leads to the development of alternative behavioral phenotypes.

Proximity-based labeling reveals DNA damage-induced phosphorylation of fused in sarcoma (FUS) causes distinct changes in the FUS protein interactome.

Accumulation of cytoplasmic inclusions containing fused in sarcoma (FUS), an RNA/DNA-binding protein, is a common hallmark of frontotemporal lobar degeneration and amyotrophic lateral sclerosis neuropathology. We have previously shown that DNA damage can trigger the cytoplasmic accumulation of N-terminally phosphorylated FUS. However, the functional consequences of N-terminal FUS phosphorylation are unknown. To gain insight into this question, we utilized proximity-dependent biotin labeling via ascorbate peroxidase 2 aired with mass spectrometry to investigate whether N-terminal phosphorylation alters the FUS protein-protein interaction network (interactome), and subsequently, FUS function. We report the first analysis comparing the interactomes of three FUS variants: homeostatic wildtype FUS (FUS WT), phosphomimetic FUS (FUS PM; a proxy for N-terminally phosphorylated FUS), and the toxic FUS proline 525 to leucine mutant (FUS P525L) that causes juvenile amyotrophic lateral sclerosis. We found that the phosphomimetic FUS interactome is uniquely enriched for a group of cytoplasmic proteins that mediate mRNA metabolism and translation, as well as nuclear proteins involved in the spliceosome and DNA repair functions. Furthermore, we identified and validated the RNA-induced silencing complex RNA helicase MOV10 as a novel interacting partner of FUS. Finally, we provide functional evidence that N-terminally phosphorylated FUS may disrupt homeostatic translation and steady-state levels of specific mRNA transcripts. Taken together, these results highlight phosphorylation as a unique modulator of the interactome and function of FUS.

Lysosome dysfunction as a cause of neurodegenerative diseases: Lessons from frontotemporal dementia and amyotrophic lateral sclerosis.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative disorders that are thought to exist on a clinical and pathological spectrum. FTD and ALS are linked by shared genetic causes (e.g. C9orf72 hexanucleotide repeat expansions) and neuropathology, such as inclusions of ubiquitinated, misfolded proteins (e.g. TAR DNA-binding protein 43; TDP-43) in the CNS. Furthermore, some genes that cause FTD or ALS when mutated encode proteins that localize to the lysosome or modulate endosome-lysosome function, including lysosomal fusion, cargo trafficking, lysosomal acidification, autophagy, or TFEB activity. In this review, we summarize evidence that lysosomal dysfunction, caused by genetic mutations (e.g. C9orf72, GRN, MAPT, TMEM106B) or toxic-gain of function (e.g. aggregation of TDP-43 or tau), is an important pathogenic disease mechanism in FTD and ALS. Further studies into the normal function of many of these proteins are required and will help uncover the mechanisms that cause lysosomal dysfunction in FTD and ALS. Mutations or polymorphisms in genes that encode proteins important for endosome-lysosome function also occur in other age-dependent neurodegenerative diseases, including Alzheimer's (e.g. APOE, PSEN1, APP) and Parkinson's (e.g. GBA, LRRK2, ATP13A2) disease. A more complete understanding of the common and unique features of lysosome dysfunction across the spectrum of neurodegeneration will help guide the development of therapies for these devastating diseases.

Divergent FUS phosphorylation in primate and mouse cells following double-strand DNA damage.

Fused in sarcoma (FUS) is a RNA/DNA protein involved in multiple nuclear and cytoplasmic functions including transcription, splicing, mRNA trafficking, and stress granule formation. To accomplish these many functions, FUS must shuttle between cellular compartments in a highly regulated manner. When shuttling is disrupted, FUS abnormally accumulates into cytoplasmic inclusions that can be toxic. Disrupted shuttling of FUS into the nucleus is a hallmark of ~10% of frontotemporal lobar degeneration (FTLD) cases, the neuropathology that underlies frontotemporal dementia (FTD). Multiple pathways are known to disrupt nuclear/cytoplasmic shuttling of FUS. In earlier work, we discovered that double-strand DNA breaks (DSBs) trigger DNA-dependent protein kinase (DNA-PK) to phosphorylate FUS (p-FUS) at N-terminal residues leading to the cytoplasmic accumulation of FUS. Therefore, DNA damage may contribute to the development of FTLD pathology with FUS inclusions. In the present study, we examined how DSBs effect FUS phosphorylation in various primate and mouse cellular models. All cell lines derived from human and non-human primates exhibit N-terminal FUS phosphorylation following calicheamicin γ1 (CLM) induced DSBs. In contrast, we were unable to detect FUS phosphorylation in mouse-derived primary neurons or immortalized cell lines regardless of CLM treatment, duration, or concentration. Despite DNA damage induced by CLM treatment, we find that mouse cells do not phosphorylate FUS, likely due to reduced levels and activity of DNA-PK compared to human cells. Taken together, our work reveals that mouse-derived cellular models regulate FUS in an anomalous manner compared to primate cells. This raises the possibility that mouse models may not fully recapitulate the pathogenic cascades that lead to FTLD with FUS pathology.

Human GNPTAB stuttering mutations engineered into mice cause vocalization deficits and astrocyte pathology in the corpus callosum.

Stuttering is a common neurodevelopmental disorder that has been associated with mutations in genes involved in intracellular trafficking. However, the cellular mechanisms leading to stuttering remain unknown. Engineering a mutation in N-acetylglucosamine-1-phosphate transferase subunits α and ß (GNPTAB) found in humans who stutter into the mouse Gnptab gene resulted in deficits in the flow of ultrasonic vocalizations similar to speech deficits of humans who stutter. Here we show that other human stuttering mutations introduced into this mouse gene, Gnptab Ser321Gly and Ala455Ser, produce the same vocalization deficit in 8-day-old pup isolation calls and do not affect other nonvocal behaviors. Immunohistochemistry showed a marked decrease in staining of astrocytes, particularly in the corpus callosum of the Gnptab Ser321Gly homozygote mice compared to wild-type littermates, while the staining of cerebellar Purkinje cells, oligodendrocytes, microglial cells, and dopaminergic neurons was not significantly different. Diffusion tensor imaging also detected deficits in the corpus callosum of the Gnptab Ser321Gly mice. Using a range of cell type-specific Cre-drivers and a Gnptab conditional knockout line, we found that only astrocyte-specific Gnptab-deficient mice displayed a similar vocalization deficit. These data suggest that vocalization defects in mice carrying human stuttering mutations in Gnptab derive from abnormalities in astrocytes, particularly in the corpus callosum, and provide support for hypotheses that focus on deficits in interhemispheric communication in stuttering.

Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-ß4-µ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the µ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering.