Biology subjects

Spitzer, M. H.

Publications and source records attributed to Spitzer, M. H..

At least 19 recordsLinked to original sources

bioRxiv2026

Programming primary human T cells to deliver gene editing machinery

Clinical applications of gene editors including CRISPR-Cas9 are limited by the cell-type specificity and tissue penetrance of current in vivo delivery options. Cell-mediated gene editor delivery could overcome these obstacles. To date, systems utilizing immortalized cell lines have demonstrated gene editor delivery in vitro. Because T cells traffic throughout the organism and engage in intercellular communication with individual target cells, we wondered whether they could be engineered to deliver gene editing machinery. Here, we demonstrate that primary human T cells can produce retroviral-based enveloped delivery vehicles (EDVs) packaging functional Cas9 ribonucleoproteins. When paired with fusogenic VSV-G, T cell-generated Cas9-EDVs edited a diverse array of target cells. We developed a miniaturized Moloney murine leukemia virus-based system that proved superior to existing architectures in this context and optimized both lentiviral and site-specific knock-in approaches for T cell engineering. These Enveloped Particle Information Courier- (EPIC-) T cells offer key advantages as delivery systems.

immunology↗

bioRxiv2026

Immune activation during broadly neutralizing antibody-mediated HIV suppression prior to post-intervention control

Broadly neutralizing antibodies (bNAbs) have been associated with enhancement of HIV-specific T or B cell responses and sustained partial control of HIV replication in some people with HIV (PWH). The mechanisms through which bNAbs may potentiate host immunity in this context are not known. We previously reported the outcomes of a clinical trial in which ten PWH on antiretroviral therapy (ART) received a combination of immunotherapies including two bNAbs administered immediately preceding an analytic treatment interruption (ATI). After bNAb levels waned, seven participants exhibited varying degrees of post-intervention control of HIV linked to a robust expansion of activated CD8+ T cells in response to rebounding virus. To investigate the role of the bNAbs in enhancing endogenous immune responses, we looked for evidence of HIV-specific or broader immune activation during the period after ART was paused and prior to rebound when bNAbs were controlling HIV replication. At a timepoint early post-ART interruption and at least one month before virus emerged in plasma, we detected an increase in levels of plasma inflammatory proteins as well as phenotypic and transcriptional activation of innate and adaptive immune cells. Compared to non-controllers, post-intervention controllers demonstrated unique transcriptional activation patterns as well as differential longitudinal plasma inflammatory protein trends. No enhancement of HIV-specific T cell or antibody responses was observed in this window. This study identifies activated cell types and inflammatory pathways that are recruited early during bNAb-mediated HIV suppression and that may play a role in potentiating long-lasting HIV immune control after bNAb therapy. One Sentence SummaryIn a combination immunotherapy trial with high rates of post-intervention control, bNAb-mediated HIV suppression was associated with increased immune activation compared to HIV suppression by ART.

immunology↗

bioRxiv2026

Successful dendritic cell vaccines require lasting in-situ TNF α secretion to license antitumor CD8 + T cell cytotoxicity

Dendritic cells (DCs) are central to activating cytotoxic CD8 T cells, yet DC-based vaccines have achieved limited success against established tumors. To address this gap, we analyzed the transcriptomic and functional changes CD8 T cells undergo following interactions with DC subsets in lymphoid organs and tumor sites. This approach allowed us to map their trajectory from naive to fully cytotoxic effector cells. We found that classical DCs in lymphoid organs provide essential antigen presentation but fail to elicit cytotoxicity. Instead, antigenexperienced CD8 T cells require additional inflammatory signals, primarily through TNF, delivered at tumor sites by infiltrating myeloid DCs. Effective cytotoxic responses therefore depend on the synchronization of these distinct, temporally separated signals. Notably, tumor antigen-pulsed DC vaccines rapidly lose TNF expression after infiltrating tumors, limiting their efficacy. These findings establish a sequential model of T cell activation and suggest strategies to enhance the potency of DC-based immunotherapies.

immunology↗

bioRxiv2026

A widespread gut bacterial lineage distinguished by redox metabolism and phage defense

Genomic variation within gut microbial species can have consequences for host health and disease. However, for low abundance species, these variations can be difficult to capture by both culture-dependent and -independent approaches. Here, we focus on the prevalent but low abundance gut Actinomycetota Eggerthella lenta. We developed a selective media for sensitive and specific isolation of E. lenta from human stool. Genomes from 87 new E. lenta isolates were combined with prior high-quality assemblies, shedding light on within-species functional diversity. Phylogenetic analysis revealed a broadly distributed subclade, which we refer to as E. lenta Group B. This lineage was differentiated by its metabolic potential and bacteriophage defense, though mobile elements were shared broadly across the species. Notably, Group B was positively associated with intestinal inflammation in subjects with inflammatory bowel disease. Overall, these results emphasize the importance of bacterial population structure in host-microbiome interactions and provide a framework to study low-abundance gut taxa. HIGHLIGHTSO_LISelective media enables E. lenta isolation and reveals high prevalence in humans C_LIO_LIDiscovery of a distinctive lineage within E. lenta undergoing genome reduction C_LIO_LIE. lenta Group B has altered metabolism, phage defense, and disease associations C_LIO_LIA widespread conjugative plasmid could enable improved genetics C_LI

microbiology↗

bioRxiv2025

IL-6 underlies microenvironment immunosuppression and resistance to therapy in glioblastoma

The glioblastoma tumor immune microenvironment (TIME) is an immunosuppressive barrier to therapy that encumbers glioblastoma responses to immune checkpoint inhibition (ICI). Immunosuppressive cytokines, pro-tumor myeloid cells, and exhausted T-cells are hallmarks of the glioblastoma TIME. Here we integrate spatial and single-cell analyses of patient-matched human glioblastoma samples before and after ICI with genetic, immunologic, single-cell, and pharmacologic studies in preclinical models to reveal that interleukin-6 (IL-6) inhibition reprograms the glioblastoma TIME to sensitize mouse glioblastoma to ICI and radiotherapy. Rare human glioblastoma patients who achieve clinical responses to ICI have lower pre-treatment IL-6 levels compared to glioblastomas who do not respond to ICI. Immune stimulatory gene therapy suppresses IL-6 tumor levels in preclinical murine models of glioblastoma. Furthermore, survival was longer in Il-6 knockout mice with orthotopic SB28 glioblastoma relative to wild-type mice. IL-6 blockade with a neutralizing antibody transiently sensitizes mouse glioblastoma to anti-PD-1 by increasing MHCII+ monocytes, CD103+ migratory dendritic cells (DCs), CD11b+ conventional DCs, and effector CD8+ T cells, and decreasing immunosuppressive Tregs. To translate these findings to a combination treatment strategy for recurrent glioblastoma patients, we show that IL-6 blockade plus ICI durably sensitizes mouse glioblastoma to high-dose radiotherapy.

cancer biology↗

bioRxiv2024

Coordinated variation in the immune systems and microbiomes of healthy humans is linked to tonic interferon states

Human immune systems are highly variable, with most variation attributable to non-genetic sources. The gut microbiome crucially shapes the immune system; however, its relationship with the baseline immune states of healthy humans remains incompletely understood. Therefore, we performed multi-omic profiling of 110 healthy participants through the ImmunoMicrobiome study. A factor-based integrative approach identified coordinated variation, revealing that the tonic interferon response was amongst the most variable immune features in healthy participants. Microbiome composition, pathways, and stool metabolites varied concomitantly with interferon response pathways. Distinct transcriptional programs involving inflammation and TGF-{beta} in SIGLEC-1high monocytes and CD69high activated MAIT and NK cells were representative of these programs. Our study provides extensive data to examine the relationship between the immune states and microbiomes of healthy individuals at steady state, which paves the way for delineating inter-individual differences relevant for disease susceptibility and responses to therapy.

immunology↗

bioRxiv2024

Lack of TGFβ signaling competency predicts immune poor cancer conversion to immune rich and response to checkpoint blockade

BackgroundThe efficacy of immune checkpoint blockade (ICB) depends on restoring immune recognition of cancer cells that have evaded immune surveillance. At the time of diagnosis, patients with lymphocyte-infiltrated cancers are the most responsive to ICB, yet a considerable fraction of patients have immune-poor tumors. MethodsWe analyzed transcriptomic data from IMvigor210, TCGA, and TISMO datasets to evaluate the predictive value of {beta}Alt, a score representing the negative correlation of signatures consisting of transforming growth factor beta (TGF{beta}) targets and genes involved in error-prone DNA repair. The immune context of {beta}Alt was assessed by evaluating tumor-educated immune signatures. An ICB-resistant, high {beta}Alt preclinical tumor model was treated with a TGF{beta} inhibitor, radiation, and/or ICB and assessed for immune composition and tumor control. ResultsHere, we show that high {beta}Alt is associated with an immune-poor context yet is predictive of ICB response in both humans and mice. A high {beta}Alt cancer in which TGF{beta} signaling is compromised generates a TGF{beta} rich, immunosuppressive tumor microenvironment. Accordingly, preclinical modeling showed that TGF{beta} inhibition followed by radiotherapy could convert an immune-poor, ICB-resistant tumor to an immune-rich, ICB-responsive tumor. Mechanistically, TGF{beta} blockade in irradiated tumors activated natural killer cells that were required to recruit lymphocytes to respond to ICB. In support of this, natural killer cell activation signatures were also increased in immune-poor mouse and human tumors that responded to ICB. ConclusionsThese studies suggest that loss of TGF{beta} competency identifies a subset of cold tumors that are candidates for ICB. Our mechanistic studies show that inhibiting TGF{beta} activity converts high {beta}Alt, cold tumors into ICB-responsive tumors via NK cells. Thus, a biomarker consisting of combined TGF{beta}, DNA repair, and immune context signatures provides a means to prospectively identify patients whose cancers may be converted from cold to hot, which could be exploited for therapeutic treatment. O_LIWhat is already known on this topic - For some cancer patients, response to ICB provides durable tumor control. Current biomarkers are insufficient to reliably predict the immunotherapy response for most patients, particularly those whose tumors lack lymphocytic infiltration. C_LIO_LIWhat this study adds - The {beta}Alt score, which reports a DNA damage deficiency caused by lack of TGF{beta} signaling, predicts response to ICB in clinical trial data from IMvigor210 metastatic bladder cancer patients and for melanoma patients. Notably, transcriptomic assessment of the immune context shows that these are immune-poor, so-called "cold" tumors. Preclinical modeling indicates that alleviating TGF{beta} inhibition of NK cells is critical to relieving immunosuppression. C_LIO_LIHow this study might affect research, practice or policy - Our work identifies a novel tumor phenotype consisting of cancer cells that have lost TGF{beta} signaling and gained error-prone DNA repair embedded in a TGF{beta} rich, immune-poor microenvironment, which is conserved across cancer types in humans and among preclinical tumor models. Patients whose immune-poor tumors have high {beta}Alt scores are strong candidates for ICB and radiotherapy combinations that may be further augmented by TGF{beta} inhibition. Hence, the {beta}Alt score can be used to stratify immune-poor cancer patients for optimal therapeutic strategies. C_LI

cancer biology↗

bioRxiv2024

Disrupting CD38-driven T cell dysfunction restores sensitivity to cancer immunotherapy

A central problem in cancer immunotherapy with immune checkpoint blockade (ICB) is the development of resistance, which affects 50% of patients with metastatic melanoma1,2. T cell exhaustion, resulting from chronic antigen exposure in the tumour microenvironment, is a major driver of ICB resistance3. Here, we show that CD38, an ecto-enzyme involved in nicotinamide adenine dinucleotide (NAD+) catabolism, is highly expressed in exhausted CD8+ T cells in melanoma and is associated with ICB resistance. Tumour-derived CD38hiCD8+ T cells are dysfunctional, characterised by impaired proliferative capacity, effector function, and dysregulated mitochondrial bioenergetics. Genetic and pharmacological blockade of CD38 in murine and patient-derived organotypic tumour models (MDOTS/PDOTS) enhanced tumour immunity and overcame ICB resistance. Mechanistically, disrupting CD38 activity in T cells restored cellular NAD+ pools, improved mitochondrial function, increased proliferation, augmented effector function, and restored ICB sensitivity. Taken together, these data demonstrate a role for the CD38-NAD+ axis in promoting T cell exhaustion and ICB resistance, and establish the efficacy of CD38 directed therapeutic strategies to overcome ICB resistance using clinically relevant, patient-derived 3D tumour models.

cancer biology↗

bioRxiv2024

A mass cytometry approach to track the evolution of T cell responses during infection and immunotherapy by paired T cell receptor repertoire and T cell differentiation state analysis

T cell receptor (TCR) recognition followed by clonal expansion is a fundamental feature of adaptive immune responses. Here, we developed a mass cytometric (CyTOF) approach combining antibodies specific for different TCR V- and V{beta}-chains with antibodies against T cell activation and differentiation proteins to identify antigen-specific expansions of T cell subsets and assess aspects of cellular function. This strategy allowed for the identification of expansions of specific V{beta} and V chain expressing CD8+ and CD4+ T cells with varying differentiation states in response to Listeria monocytogenes, tumors, and respiratory influenza infection. Expanded V{beta} chain expressing T cells could be directly linked to the recognition of specific antigens from Listeria, tumor cells, or influenza. In the setting of influenza infection, we showed that the common therapeutic approaches of intramuscular vaccination or convalescent serum transfer altered the clonal diversity and differentiation state of responding T cells. Thus, we present a new method to monitor broad changes in TCR specificity paired with T cell differentiation during adaptive immune responses.

immunology↗

bioRxiv2023

Enhancing CAR-T Cell Metabolism to Overcome Hypoxic Conditions in the Brain Tumor Microenvironment

The efficacy of chimeric antigen receptor (CAR)-T therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28-EGFRvIII glioma revealed impaired mitochondrial ATP production and a markedly hypoxic status compared to ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of AMPK activator Metformin and the mTOR inhibitor Rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-gamma coactivator 1 (PGC-1) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective anti-glioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28-EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group with fewer Ly6c+ CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.

immunology↗

bioRxiv2023

Commensal myeloid crosstalk in neonatal skin regulates long-term cutaneous type 17 inflammation

Early life microbe-immune interactions at barrier surfaces have lasting impacts on the trajectory towards health versus disease. Monocytes, macrophages and dendritic cells are primary sentinels in barrier tissues, yet the salient contributions of commensal-myeloid crosstalk during tissue development remain poorly understood. Here, we identify that commensal microbes facilitate accumulation of a population of monocytes in neonatal skin. Transient postnatal depletion of these monocytes resulted in heightened IL-17A production by skin T cells, which was particularly sustained among CD4+ T cells and sufficient to exacerbate inflammatory skin pathologies. Neonatal skin monocytes were enriched in expression of negative regulators of the IL-1 pathway. Functional in vivo experiments confirmed a key role for excessive IL-1R1 signaling in T cells as contributing to the dysregulated type 17 response in neonatal monocyte-depleted mice. Thus, a commensal-driven wave of monocytes into neonatal skin critically facilitates immune homeostasis in this prominent barrier tissue.

immunology↗

bioRxiv2022

Epigenetic reprogramming shapes the cellular landscape of schwannoma

Cell state evolution underlies tumor development and response to therapy1, but mechanisms specifying cancer cell states and intratumor heterogeneity are incompletely understood. Schwannomas are the most common tumors of the peripheral nervous system and are treated with surgery and ionizing radiation2-5. Schwannomas can oscillate in size for many years after radiotherapy6,7, suggesting treatment may reprogram schwannoma cells or the tumor microenvironment. Here we show epigenetic reprogramming shapes the cellular landscape of schwannomas. We find schwannomas are comprised of 2 molecular groups distinguished by reactivation of neural crest development pathways or misactivation of nerve injury mechanisms that specify cancer cell states and the architecture of the tumor immune microenvironment. Schwannoma molecular groups can arise independently, but ionizing radiation is sufficient for epigenetic reprogramming of neural crest to immune-enriched schwannoma by remodeling chromatin accessibility, gene expression, and metabolism to drive schwannoma cell state evolution and immune cell infiltration. To define functional genomic mechanisms underlying epigenetic reprograming of schwannomas, we develop a technique for simultaneous interrogation of chromatin accessibility and gene expression coupled with genetic and therapeutic perturbations in single-nuclei. Our results elucidate a framework for understanding epigenetic drivers of cancer evolution and establish a paradigm of epigenetic reprograming of cancer in response to radiotherapy.

cancer biology↗

bioRxiv2022

Dynamic CD8+ T cell responses to cancer immunotherapy in human regional lymph nodes are disrupted by metastasis

CD8+ T cell responses are critical for anti-tumor immunity. While extensively profiled in the tumor microenvironment (TME), recent studies in mice identified responses in lymph nodes (LN) as essential; however, the role of LN in human cancer patients remains unknown. We examined CD8+ T cells in human head and neck squamous cell carcinomas, regional LN, and blood using mass cytometry, single-cell genomics, and multiplexed ion beam imaging. We identified progenitor exhausted CD8+ T cells (Tpex) that were abundant in uninvolved LN and clonally related to terminally exhausted cells in the TME. After anti-PD-L1 immunotherapy, Tpex in uninvolved LN reduced in frequency but localized near dendritic cells and proliferating intermediate-exhausted CD8+ T cells (Tex-int), consistent with activation and differentiation. LN responses coincided with increased circulating Tex-int. In metastatic LN, these response hallmarks were impaired by immunosuppressive cellular niches. Our results identify important roles for LN in anti-tumor immune responses in humans.

immunology↗

bioRxiv2022

T cell-instructed inflammation drives immune checkpoint inhibitor therapy resistance

Resistance to immune checkpoint inhibitors (ICIs) is common, even in tumors with T cell infiltration. We thus investigated consequences of ICI-induced T cell infiltration in the microenvironment of resistant tumors. T cells increased in ICI-resistant tumors following treatment as did neutrophils, in contrast to ICI-responsive tumors. Resistant tumors were distinguished by high expression of IL-1 Receptor 1 (IL1R1), enabling a synergistic response to IL-1 and TNF to induce G-CSF, CXCL1, and CXCL2 via NF-{kappa}B signaling, supporting neutrophils. Perturbation of this inflammatory resistance circuit sensitized tumors to ICIs. Paradoxically, T cells drove this resistance circuit via TNF both in vitro and in vivo. Evidence of this inflammatory resistance circuit and its impact also translated to human cancers. These data support a novel mechanism of ICI resistance, wherein treatment-induced T cell activity can drive resistance in tumors responsive to IL-1 and TNF, with important therapeutic implications. Statement of SignificanceAlthough T cell-infiltrated cancers are frequently resistant to immune checkpoint inhibitor therapies, mechanisms of resistance beyond T cell exhaustion remain unclear. Here, we reveal the functional significance of tumor- infiltrating T cells in resistant tumors, which surprisingly instruct immunosuppressive inflammation in mouse and human cancers responsive to IL-1 and TNF.

immunology↗

bioRxiv2022

A conserved immune trajectory of recovery in hospitalized COVID-19 patients

Many studies have provided insights into the immune response to COVID-19; however, little is known about the immunological changes and immune signaling occurring during COVID-19 resolution. Individual heterogeneity and variable disease resolution timelines obscure unifying immune characteristics. Here, we collected and profiled >200 longitudinal peripheral blood samples from patients hospitalized with COVID-19, with other respiratory infections, and healthy individuals, using mass cytometry to measure immune cells and signaling states at single cell resolution. COVID-19 patients showed a unique immune composition and an early, coordinated and elevated immune cell signaling profile, which correlated with early hospital discharge. Intra-patient time course analysis tied to clinically relevant events of recovery revealed a conserved set of immunological processes that accompany, and are unique to, disease resolution and discharge. This immunological process, together with additional changes in CD4 regulatory T cells and basophils, accompanies recovery from respiratory failure and is associated with better clinical outcomes at the time of admission. Our work elucidates the biological timeline of immune recovery from COVID-19 and provides insights into the fundamental processes of COVID-19 resolution in hospitalized patients.

immunology↗

bioRxiv2021

Transient dendritic cell activation diversifies the T cell response to acute infection

The precise timing of T cell priming during infection remains unclear. Here, we mapped the cellular dynamics of all immune lineages during acute infection with Listeria monocytogenes (Lm). We identified highly transient DC activation 2 days post-infection that functions as a critical time window for priming effector T cells. Regulation of this transient state was mediated by DC extrinsic IFN{gamma} provided by lymphocytes. Furthermore, antigen-specific T cells that arrive late to the site of priming and miss peak DC activation acquire only memory T cell fates. This temporal regulation of fate is recapitulated by CD8+ DCs ex vivo, suggesting that shifts in activation state of a single antigen presenting cell population alter T cell fates. These results uncover a novel mechanism for temporal regulation of T cell differentiation during a dynamic immune response to acute infection. One-Sentence SummaryThe immune system generates a balanced protective response through rapidly shifting activation states and recruitment of new cells into an ongoing inflammatory landscape.

immunology↗

bioRxiv2021

Distinct cellular immune signatures in acute Zika virus infection are associated withhigh or low persisting neutralizing antibody titers

Although the formation of a durable neutralizing antibody response after an acute viral infection is a key component of protective immunity, little is known about why some individuals generate high versus low neutralizing antibody titers to infection or vaccination. Infection with Zika virus (ZIKV) during pregnancy can cause devastating fetal outcomes, and efforts to understand natural immunity to this infection are essential for optimizing vaccine design. In this study, we leveraged the high-dimensional single-cell profiling capacity of mass cytometry (CyTOF) to deeply characterize the cellular immune response to acute and convalescent ZIKV infection in a cohort of blood donors in Puerto Rico incidentally found to be viremic during the 2015-2016 epidemic in the Americas. During acute ZIKV infection, we identified widely coordinated responses across innate and adaptive immune cell lineages. High frequencies of multiple activated innate immune subsets, as well as activated follicular helper CD4+ T cells and proliferating CD27-IgD-B cells, during acute infection were associated with high titers of ZIKV neutralizing antibodies at 6 months post-infection. On the other hand, low titers of ZIKV neutralizing antibodies were associated with immune features that suggested a cytotoxic-skewed immune "set-point." Our study offers insight into the cellular coordination of immune responses and identifies candidate cellular biomarkers that may offer predictive value in vaccine efficacy trials for ZIKV and other acute viral infections aimed at inducing high titers of neutralizing antibodies. One Sentence SummaryMass cytometry reveals acute ZIKV infection cellular immune signatures that predict high or low neutralizing antibody titers 6 months post-infection.

immunology↗

bioRxiv2021

Type 2 innate immunity regulates hair follicle homeostasis to control Demodex pathosymbionts

Allergic skin diseases are common, but basal roles for type 2 immunity in cutaneous homeostasis are incompletely understood. Here, we show that skin group 2 innate lymphoid cells (ILC2s) are the predominant resident cells that secretes IL-13, which attenuates epithelial cell proliferation during anagen throughout the hair follicle (HF), including in HF stem cells. Although HF are normal in the absence of type 2 immunity, colonization with the commensal mite, Demodex musculi, results in epithelial proliferation and aberrant hair follicle morphology accompanied by loss of stable commensalism with massive infestation and expansion of inflammatory ILC2s and other immune cells. Topical anti-parasitic agents, but not broad-spectrum antibiotics, reversed the phenotype. Commensal Demodex colonization of mammalian hair follicles is ubiquitous, including in humans, revealing an unanticipated role for ILC2s and type 2 immunity for skin homeostasis in the normal environment. One Sentence SummaryDynamic regulation of skin physiology by Type 2 innate immunity regulates healthy commensalism by parasitic Demodex mites.

immunology↗