Biology subjects

Castillo, J. G.

Publications and source records attributed to Castillo, J. G..

9 recordsLinked to original sources

bioRxiv2025

MAIT cells induced by engineered Listeria exhibit antibacterial and antitumor activity

Mucosal-associated invariant T (MAIT) cells are among the most conserved and abundant innate-like T cells in humans that recognize microbial-derived riboflavin precursors and elicit potent antimicrobial responses. The foodborne pathogen Listeria monocytogenes is a broad host-range facultative intracellular pathogen that lacks the riboflavin biosynthetic pathway, leading us to hypothesize that this deficiency is pathoadaptive and allows the pathogen to evade MAIT cells. Here, we show that L. monocytogenes strains engineered to produce riboflavin (L. monocytogenes-ribDEAHT) are attenuated in wild-type mice but fully virulent in MAIT cell-deficient mice. Infection with L. monocytogenes-ribDEAHT prompted rapid and robust MAIT cell expansion in multiple tissues and required the cytolytic effector perforin to eliminate infected cells in vivo and in vitro. We also assessed the therapeutic potential of L. monocytogenes-ribDEAHT-stimulated MAIT cells in both infectious disease and cancer mouse models. Therapeutic administration of L. monocytogenes-ribDEAHT provided protection against Francisella tularensis in the lungs and inhibited tumor growth even in the absence of CD8+ T cells. These findings reveal the importance of MAIT cell evasion during L. monocytogenes infection and highlight the therapeutic potential of engineered L. monocytogenes to activate and harness MAIT cells for protection against infectious disease and cancer. Significance StatementListeria monocytogenes is a bacterial pathogen that grows freely in the environment but can become intracellular following ingestion of contaminated food. Although L. monocytogenes can synthesize most metabolites required for growth, it lacks the genes necessary to produce riboflavin (vitamin B2), an essential cofactor across all domains of life. We hypothesized that lacking riboflavin biosynthesis allows L. monocytogenes to evade mucosal-associated invariant T cells (MAIT cells), which generate potent antimicrobial responses against riboflavin-producing microbes. By engineering L. monocytogenes to produce riboflavin, we show that these strains robustly activate MAIT cells and are highly attenuated in wild-type mice, but not in MAIT cell-deficient mice. Furthermore, MAIT cells activated by engineered L. monocytogenes provided therapeutic protection against other riboflavin-producing bacteria and cancer.

microbiology↗

bioRxiv2025

Selective disruption of lipid peroxide homeostasis in intratumoral regulatory T cells by targeting FSP1 enhances cancer immunity

A burgeoning approach to treat cancer is the pharmacological induction of ferroptotic cell death of tumor cells. However, the impact of disrupting anti-ferroptotic pathways in the broader tumor microenvironment (TME), such as in immune cells, is still undefined and may complicate treatments. Here, we show that Ferroptosis Suppressor Protein 1 (FSP1/Aifm2) is critically required for regulatory T cell (Treg) resistance to ferroptosis and their immunosuppressive function within the TME. Compared to other canonical ferroptosis regulators such as GPX4, GCH1, and NRF2, only FSP1 was induced upon T cell activation. Deletion of Aifm2 in all T cells, or Tregs specifically, enhanced tumor control by selectively disrupting Treg immunosuppression within tumors without inciting autoimmune pathology in mice. As opposed to deletion of Gpx4 in all T cells, T cell deletion of Aifm2 did not impair antigen-specific CD8+ T cell responses. These results reveal a unique opportunity for targeting a regulator of ferroptosis that can not only directly target cancer cells, but also simultaneously enhance anti-cancer immune responses without inciting autoimmunity.

immunology↗

bioRxiv2025

TLR2 signaling uniquely destabilizes tumor Tregs to promote cancer immunotherapy

A fundamental principle of immune responses is that innate immunity promotes adaptive immunity, but in the context of cancer immunity, the importance of innate receptor signaling is poorly defined. To study how Toll-like receptor (TLR) signaling contributes to cancer immunity, we used an attenuated strain of Listeria monocytogenes (Lm) that inhibits tumor growth in mice. We found that Lm stimulation of TLR2, but not TLR5 or TLR9, was essential for tumor control. As expected, TLR2 supported the priming of Lm-specific T cells in lymphoid tissues. However, TLR2 signaling in innate immune cells within tumors also destabilized Foxp3 expression in regulatory T cells (Tregs), a function that was not mediated by other TLRs. Reduced Treg function promoted tumor antigen cross-presentation by DC1s to enhance the functionality of recalled tumor-specific CD8+ T cells directly within tumors. These findings reveal a unique capacity for TLR2 signaling to diminish immunosuppression and promote cancer immunity.

immunology↗

bioRxiv2024

Age-related remodeling of the glycocalyx drives T cell exhaustion

Cell surface glycans, termed the glycocalyx, are essential regulators of cellular signaling and thus cellular development and functions, but how aging impacts the glycocalyx remains poorly understood. Here, using immune cells as a model system for studying the relationship between aging and glycocalyx remodeling, we show that 2,6-linked sialic acid - a terminal glycan epitope typically associated with inhibitory signaling - becomes downregulated in T cells from older animals. This downregulation is tightly correlated with age-associated accumulation of effector T cells, which are decorated with little to no 2,6-linked sialic acids. T cell aging renders older individuals more vulnerable to infections and cancers. To understand the role of 2,6-linked sialic acids in T cell physiology, we generated a mouse model with T cell-specific deletion of the sialyltransferase gene St6gal1. The chronic depletion of 2,6-linked sialic acids led to naive T (TN) cells expansion in the periphery and premature T cell exhaustion. As a result, these mice were less able to control acute Listeria infection and chronic tumor growth. Blockade of the PD-1 pathway can partially restore the ability of St6gal1-deficient T cells to control tumor growth. Together, these data suggest that 2,6-linked sialic acids are critical for maintaining long-term T cell responsiveness, and the loss of 2,6-linked sialic acids may directly contribute to age-related T cell exhaustion.

immunology↗

bioRxiv2024

Cellular mechanisms underlying beneficial versus detrimental effects of bacterial antitumor immunotherapy

Bacteria engineered to express tumor antigens as a cancer vaccine have yielded mixed results. Here, we utilized an attenuated strain of Listeria monocytogenes ({Delta}actA, Lm) that does not express tumor antigen to explore the immunological response to Listeria itself in the context of intravenous (IV), intratumoral (IT), or a combination of IV+IT administration into tumor-bearing mice. Unexpectedly, we found that Lm persisted in tumors of immune competent mice, regardless of the administration route. While IT Lm alone led to the recruitment of immunosuppressive immune cells that promoted tumor growth, IV Lm followed by IT Lm controlled tumor growth. IV Lm vaccination generated a pool of anti-Lm cytotoxic CD8 T cells that killed Lm-infected non-tumor cells to control tumor growth both indirectly, by limiting cancer cell proliferation, and directly, by enhancing tumor-specific T cell responses. Our findings reveal a differential impact of IT Lm administration on tumor progression that depends on the presence of anti-Lm CD8 T cells, which alone are sufficient to promote therapeutic efficacy.

immunology↗

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↗

bioRxiv2021

Aberrant FGF signaling promotes granule neuron precursor expansion in SHH subgroup infantile medulloblastoma

Mutations in Sonic Hedgehog (SHH) signaling pathway genes, e.g., Suppressor of Fused (SUFU), drive granule neuron precursors (GNP) to form medulloblastomas (MBSHH). However, how different molecular lesions in the Shh pathway drive transformation is frequently unclear, and SUFU mutations in the cerebellum seem distinct. In this study, we show that fibroblast growth factor 5 (FGF5) signaling is integral for many infantile MBSHH cases and that FGF5 expression is uniquely upregulated in infantile MBSHH tumors. Similarly, mice lacking SUFU (Sufu-cKO) ectopically express Fgf5 specifically along the secondary fissure where GNPs harbor preneoplastic lesions and show that FGFR signaling is also ectopically activated in this region. Treatment with an FGFR antagonist rescues the severe GNP hyperplasia and restores cerebellar architecture. Thus, direct inhibition of FGF signaling may be a promising and novel therapeutic candidate for infantile MBSHH.

cancer biology↗

bioRxiv2018

Suppressor of Fused controls perinatal expansion and quiescence of future dentate adult neural stem cells

Adult hippocampal neurogenesis requires the quiescent neural stem cell (NSC) pool to persist lifelong. The establishment and maintenance of quiescent NSC pools during development is not understood. Here we show that Suppressor of Fused (Sufu) controls establishment of the quiescent NSC pool during mouse dentate gyrus (DG) development through regulation of Sonic Hedgehog (Shh) signaling. Deletion of Sufuin NSCs at the beginning of DG development decreases Shh signaling leading to reduced proliferation of NSCs, resulting in a small quiescent NSC pool in adult mice. We found that putative adult NSCs proliferate and increase their numbers in the first postnatal week and subsequently become quiescent towards the end of the first postnatal week. In the absence of Sufu, postnatal expansion of NSCs is compromised, and NSCs prematurely become quiescent. Thus, Sufu is required for Shh signaling activity ensuring the expansion and proper transition of NSC pools to quiescence during DG development.

developmental biology↗

bioRxiv2018

Suppressor of Fused controls the proliferation of postnatal neural stem and precursor cells via a Gli3-dependent mechanism

The ventricular-subventricular zone (V-SVZ) of the forebrain is the source of neurogenic stem/precursor cells for adaptive and homeostatic needs throughout the life of most mammals. Here, we report that Suppressor of Fused (SUFU) plays a critical role in the establishment of the V-SVZ at early neonatal stages by controlling the proliferation of distinct subpopulations of stem/precursor cells. Conditional deletion of Sufu in radial glial progenitor cells (RGCs) at E13.5 resulted in a dramatic increase in the proliferation of Sox2+ Type B cells, as well as Gsx2+ ventral forebrain derived transit amplifying precursor cells (TACs). In contrast, we found a significant decrease in Tbr2+ dorsal forebrain derived TACs indicating that innate differences between dorsal and ventral forebrain derived Type B cells influence Sufu function. However, most precursors failed to survive and accumulated in the dorsal V-SVZ, demonstrating that precursors are unable to transition into functional differentiated progenies. These defects were accompanied by reduced Gli3 expression, yet despite reduced Gli3 levels, activation of Sonic hedgehog (Shh) signaling did not occur implying that the Sufu-Gli3 regulatory axis may influence other signaling pathways in the neonatal dorsal V-SVZ. These data suggest that Sufu plays a critical role in controlling Gli3 function in the establishment and survival of functional stem/precursor cells in the postnatal dorsal V-SVZ.\n\nSUMMARY STATEMENTConditional deletion of Sufu cause dramatic expansion of neural stem/precursor cells in the neonatal ventricular-subventricular (SVZ) zone. This defect occurs through a Gli3-dependent mechanism that results in the downregulation of Shh signaling.

developmental biology↗