Biology subjects

Spitzer, M.

Publications and source records attributed to Spitzer, M..

7 recordsLinked to original sources

bioRxiv2025

Coordinated macrophage and T cell interactions mediate response to checkpoint blockade in colorectal cancer.

Mismatch repair deficiency (MMRd), either due to inherited or somatic mutation, is prevalent in colorectal cancer (CRC) and other cancers. While anti-PD-1 therapy is utilized in both local and advanced disease, up to 50% of MMRd CRC fail to respond. Using animal and human models of MMRd, we determined that interactions between MHC+ C1Q+ CXCL9+ macrophages and TCF+ BHLHE40+ PRF1+ T cell subsets are associated with control of MMRd tumor growth, during anti-PD-1 treatment. In contrast, resistance is associated with upregulation of TIM3, LAG3, TIGIT, and PD-1 expression on T cells, and infiltration of the tumor with immunosuppressive TREM2+ macrophages and monocytes. By combining anti-PD-1 with anti-LAG3/CTLA4/TREM2, up to 100% tumor eradication was achieved in MMRd CRC and remarkably, in >70% in MMRp CRC. This study identifies key T cell and macrophage subsets mediating the efficacy of immunotherapy in overcoming immune escape in both MMRd and MMRp CRC settings. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/637954v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1778bd6org.highwire.dtl.DTLVardef@17da4e5org.highwire.dtl.DTLVardef@1d4d1d2org.highwire.dtl.DTLVardef@11b58e3_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAnti-PD-1 therapy leads to the accumulation and colocalization of MHCI/II+ C1Q+ CXCL9+ macrophages and DCs with TCF+ CCL5+ T cells that have high TCR diversity. C_LIO_LIResistance to anti-PD-1 therapy involves multiple T cell checkpoints, TREM2+ macrophages, IL1B+ TREM1+ monocytes and neutrophils, and IFITM+ tumor cells. C_LIO_LISimultaneous blockade of PD-1, LAG3, CTLA-4 and TREM2 dramatically prevents progression of both MMRd and MMRp tumors. C_LIO_LICombination therapy completely eliminates tumors by leveraging MHC+ macrophage, CD4+ and CD8+ T cell interactions, facilitating durable anti-tumor effects. C_LI

immunology↗

bioRxiv2025

Butyrolactol A is a phospholipid flippase inhibitor that potentiates the bioactivity of caspofungin against resistant fungi

Fungal infections cause millions of deaths annually and are challenging to treat due to limited therapeutic options and rising resistance. Cryptococci are intrinsically resistant to the latest generation of antifungals, echinocandins, while Candida auris, a notorious global threat, is also increasingly resistant. We performed a natural product extract screen to rescue caspofungin fungicidal activity against Cryptococcus neoformans H99 and identified butyrolactol A, which restores echinocandin efficacy against resistant fungal pathogens, including multidrug-resistant C. auris. Mode of action studies revealed that butyrolactol A inhibits the phospholipid flippase Apt1-Cdc50, blocking phospholipid transport. Cryo-electron microscopy analysis of the Apt1*butyrolactol A complex revealed that the flippase is trapped in a dead-end state. Apt1 inhibition disrupts membrane asymmetry, vesicular trafficking, and cytoskeletal organization, thereby enhancing echinocandin uptake and potency. This study identifies lipid flippases as promising antifungal targets and demonstrates the potential of revisiting natural products to expand the antifungal arsenal and combat resistance.

biochemistry↗

bioRxiv2023

Single cell proteomics characterization of bone marrow hematopoiesis with distinct Ras pathway lesions.

Normal hematopoiesis requires constant prolific production of different blood cell lineages by multipotent hematopoietic stem cells (HSC). Stem- and progenitor- cells need to balance dormancy with proliferation. How genetic alterations impact frequency, lineage potential, and metabolism of HSC is largely unknown. Here, we compared induced expression of KRASG12D or RasGRP1 to normal hematopoiesis. At low-resolution, both Ras pathway lesions result in skewing towards myeloid lineages. Single-cell resolution CyTOF proteomics unmasked an expansion of HSC- and progenitor- compartments for RasGRP1, contrasted by a depletion for KRASG12D. SCENITH quantitates protein synthesis with single-cell precision and corroborated that immature cells display low metabolic SCENITH rates. Both RasGRP1 and KRASG12D elevated mean SCENITH signals in immature cells. However, RasGRP1-overexpressing stem cells retain a metabolically quiescent cell-fraction, whereas this fraction diminishes for KRASG12D. Our temporal single cell proteomics and metabolomics datasets provide a resource of mechanistic insights into altered hematopoiesis at single cell resolution.

developmental biology↗

bioRxiv2023

The lncRNA Malat1 Inhibits miR-15/16 to Enhance Cytotoxic T Cell Activation and Memory Cell Formation

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, many intracellular bacteria and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 also play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and T cell memory. Comparative Argonaute-2 high throughput sequencing of crosslinking immunoprecipitation (Ago2 HITS-CLIP, or AHC) combined with gene expression profiling in normal and miR-15/16-deficient T cells revealed a large network of several hundred direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, the long non-coding RNA Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak in T cells. This binding site was also among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16 binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of IL-2 and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long noncoding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=197 SRC="FIGDIR/small/536843v2_figu1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1ebb1bborg.highwire.dtl.DTLVardef@e19c29org.highwire.dtl.DTLVardef@1d3568forg.highwire.dtl.DTLVardef@1d7d69b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2019

Immune disease variants modulate gene expression in regulatory CD4+ T cells and inform drug targets

Identifying cellular functions dysregulated by disease associated variants could implicate novel pathways for drug targeting or modulation in cell therapies. Variants associated with immune diseases point towards the role of CD4+ regulatory T cells (Tregs), cell type critical for immune homeostasis. To translate the effects of immune disease alleles on Treg function we mapped genetic regulation (QTL) of gene expression and chromatin activity in Tregs. We identified 123 loci where Treg QTLs colocalized with immune disease variants. These colocalizations indicated that dysregulation of key Treg pathways, including cell activation via CD28 and IL-2 signalling mediated by STAT5A contribute to the shared pathobiology of immune diseases. Finally, using disease GWAS signals colocalizing with Treg QTLs, we identified 34 known drug targets and 270 targets with drug tractability evidence. Our study is the first in-depth characterization of immune disease variant effects on Treg gene expression modulation and dysregulation of Treg function.

genetics↗

bioRxiv2018

Identification of RNA-associated peptides, iRAP, defines precise sites of protein-RNA interaction

ABSRACTThe identification of ever-increasing numbers of RNA species has underlined the importance of robust characterization of bona fide sites of protein-RNA interaction. UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. Here we sought to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, identification of RNA-associated peptides (iRAP), was used to locate 1331 unique RNA-interaction sites at single amino acid residue resolution in 324 S. cerevisiae proteins. Our identified RNA-interaction sites in characterized RNA-protein complex agree well with available high-resolution structures. In known RNA-interacting protiens, 317 sites fall into known and suspected RNA-interaction domains while only 21 sites fall into other annotated sequence features. Strikingly, 993 of the sites identified fall into protein regions that lack any recognizable protein domain structure or annotated sequence features. This suggests that, despite binding RNA in vivo, many of these proteins will not have defined functions in RNA metabolism.\n\nHighlights Method identifies precise RNA-binding sites across proteome\n1331 sites identified that contact RNA in 324 proteins in S. cerevisiae\nRNA binding takes place predominantly outside known protein domains

molecular biology↗

bioRxiv2018

Depletion of microbiome-derived molecules in the host using Clostridium genetics

The gut microbiota produce hundreds of molecules that are present at high concentrations in circulation and whose levels vary widely among humans. In most cases, molecule production has not been linked to specific bacterial strains or metabolic pathways, and unraveling the contribution of each molecule to host biology remains difficult. A general system to toggle molecules in this pool on/off in the host would enable interrogation of the mechanisms by which they modulate host biology and disease processes. Such a system has been elusive due to limitations in the genetic manipulability of Clostridium and its relatives, the source of many molecules in this pool. Here, we describe a method for reliably constructing clean deletions in a model commensal Clostridium, C. sporogenes (Cs), including multiply mutated strains. We demonstrate the utility of this method by using it to toggle off the production of ten Cs-derived molecules that accumulate in host tissues. By comparing mice colonized by wild-type Cs versus a mutant deficient in the production of branched short-chain fatty acids, we discover a previously unknown IgA-modulatory activity of these abundant microbiome-derived molecules. Our method opens the door to interrogating and sculpting a highly concentrated pool of chemicals from the microbiome.

microbiology↗