Biology subjects

Bowen, J. R.

Publications and source records attributed to Bowen, J. R..

6 recordsLinked to original sources

bioRxiv2026

Engineering antigen-driven co-stimulation and T helper cell activity into TCR-T cells with CD8-41BB fusion receptors enhances anti-tumor activity

1Adoptive cell therapy using tumor antigen-targeting T cell receptors (TCRs) offers a compelling approach to treat both hematological cancers and solid tumors due to broad antigen accessibility and the ability to target cancer-specific neoantigens. However, unlike clinically validated second generation CAR-T cells bearing built-in co-stimulatory signaling modules (i.e. 41BB or CD28), TCR-T cells receive little to no co-stimulation within most tumor microenvironments leading to attenuated cellular responses. Additionally, CD4+ TCR-T cells engineered to express HLA-Class I restricted TCRs possess minimal T-helper cell activity and thus do not effectively mobilize CD8+ TCR-T cells or host anti-tumor immune responses. To address these limitations, we used CRISPR-Cas9 to engineer TCR-T cells with targeted integration of chimeric CD8 constructs containing intracellular co-stimulatory domains. We found that expression of wild-type CD8{beta}, but not CD8, could promote CD4+ T cell activities in HLA-Class I restricted TCR-T cells. However, this was insufficient to drive durable anti-tumor responses in challenging tumor mouse models when using a high-affinity WT1-directed TCR. To address this, several CD8 co-stimulatory fusion constructs containing CD28 or 41BB intracellular domains were designed and screened, identifying two CD8-41BB based chimeras that substantially increased TCR-T cell activity relative to wild-type CD8{beta}. WT1-TCR-T cells co-expressing the CD8-41BB fusions demonstrated not only enhanced CD4+ activity including strong and polarized Th1-type cytokine secretion, but also enhanced the proliferation, cytokine release, and cytotoxicity of CD8+ CTLs. Remarkably, when combined with TGFBR2 gene disruption, WT1-TCR-T cells co-expressing CD8-41BB receptors were able to completely regress established cell line-derived ovarian tumors, showed robust in vivo expansion and persistence, and provided long-term protection from tumor rechallenge. Importantly, the specificity profile of the WT1-TCR including its HLA-A*02:01 restriction and WT1 peptide recognition motif was preserved upon expression of CD8-41BB. To simplify cell engineering processes for clinical applications, we configured a homology directed repair (HDR) cassette to allow for efficient CRISPR-Cas9-based insertion of both the TCR and CD8-41BB transgenes in the TRAC locus in a single step with >80% efficiency. Lastly, the enhanced activity conferred by CD8-41BB expression was validated with a second clinically relevant TCR targeting PRAME, suggesting this platform can be a universal approach for enhancing the therapeutic potential of TCR-based cell therapies.

synthetic biology↗

bioRxiv2022

Inhibition of CHK2 dependent DNA damage response suppresses Zika virus infection through a STING dependent mechanism

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes neurological disorders and microcephaly. Recent research has shown that ZIKV causes cell cycle arrest and DNA damage response in neuronal progenitor cells that potentially leads to congenital neurodevelopmental defects. However, the specific role of regulators that control DNA damage response to ZIKV infection and the related mechanisms remain largely unknown. Here through both in vitro and in vivo studies, we observe that ZIKV induces DNA damage response in both human cell line and mouse embryo. When CHK2 dependent DNA damage response pathway is inhibited by a small molecule inhibitor or genetic deletion, ZIKV production is reduced and the embryonic developmental defects are rescued. Furthermore, we demonstrate that in ZIKV infected Chk2-/- mice, the reduced viral load correlates with elevated antiviral innate immune response which is found to be dependent on the STING signaling pathway. Collectively, our study reveals a specific role of CHK2 for ZIKV infection and pathogenesis, demonstrating a mechanism that inhibition of the CHK2 axis suppresses ZIKV infection through the STING-dependent antiviral pathway, thus providing a new therapeutic strategy against ZIKV. These findings also suggest the intriguing possibility that ZIKV evolved to orchestrate DNA damage response factors to create a beneficial environment for its infection in the host cells.

immunology↗

bioRxiv2020

Activity-based urinary biomarkers of response and resistance to checkpoint blockade immunotherapy

Immune checkpoint blockade (ICB) therapy has transformed cancer treatment, yet most patients do not derive clinical benefit and responders can acquire resistance to therapy. Noninvasive biomarkers are needed to indicate early on-treatment response and resistance mechanisms. Here we developed ImmuNe Sensors for monItorinG cHeckpoint blockade Therapy (INSIGHT), which comprises a library of mass-barcoded peptide substrates conjugated to PD1 antibodies, as therapeutic sensors. Following systemic administration, INSIGHT carries out the dual role of reinvigorating T cell function and profiling T cell and tumor proteases by the release of cleaved peptides into urine for noninvasive detection by mass spectrometry. We show that an PD1 therapeutic sensor for Granzyme B discriminates early treatment responses before tumor volumes significantly diverge from isotype controls in murine models of colorectal cancer. To differentiate mechanisms of resistance by multivariate analysis, we design a mass-barcoded, 14-plex INSIGHT library to profile proteases differentially expressed by tumors harboring B2m or Jak1 loss-of-function mutations. We find that binary classifiers trained on urine samples indicate response to PD-1 therapy as early as the start of the second dose, and discriminate B2m from Jak1 resistance with high sensitivity and specificity (AUROCs > 0.9). Our data supports the use of activity-based biomarkers for early on-treatment response assessment and classification of refractory tumors based on resistance mechanisms. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/420265v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@12ce799org.highwire.dtl.DTLVardef@118fcc5org.highwire.dtl.DTLVardef@17b227eorg.highwire.dtl.DTLVardef@115a7fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

STAT5: A Target of Antagonism by Neurotropic Flaviviruses

Flaviviruses are a diverse group of arthropod-borne viruses responsible for numerous significant public health threats; therefore, understanding the interactions between these viruses and the human immune response remains vital. Earlier work has found that WNV and ZIKV infect human DCs and can block antiviral immune responses in DCs. Previously, we used mRNA sequencing and weighted gene co-expression network analysis (WGCNA) to define molecular signatures of antiviral DC responses following activation of innate immune signaling (RIG-I, MDA5, or type I IFN signaling) or infection with WNV. Using this approach, we found that several genes involved in T cell co-signaling and antigen processing were not enriched in DCs during WNV infection. Using cis-regulatory sequence analysis, STAT5 was identified as a regulator of DC activation and immune responses downstream of innate immune signaling that was not activated during either WNV or ZIKV infection. Mechanistically, WNV and ZIKV actively blocked STAT5 phosphorylation downstream of RIG-I, IFN{beta}, and IL-4, but not GM-CSF signaling. Unexpectedly, dengue virus serotypes 1-4 (DENV1-4) and the yellow fever 17D vaccine strain (YFV-17D) did not antagonize STAT5 phosphorylation. In contrast to WNV, ZIKV inhibited JAK1 and TYK2 phosphorylation following type I IFN treatment, suggesting divergent mechanisms used by these viruses to inhibit STAT5 activation. Combined, these findings identify STAT5 as a target of antagonism by specific pathogenic flaviviruses to subvert the immune response in infected DCs.\n\nImportanceFlaviviruses are a diverse group of insect-borne viruses responsible for numerous significant public health threats. Previously, we used a computational biology approach to define molecular signatures of antiviral DC responses following activation of innate immune signaling or infection with WNV. In this work, we identify STAT5 as a regulator of DC activation and antiviral immune responses downstream of innate immune signaling that was not activated during either WNV or ZIKV infection. WNV and ZIKV actively blocked STAT5 phosphorylation downstream of RIG-I, IFN{beta}, and IL-4, but not GM-CSF signaling. However, other related flaviviruses, dengue virus serotypes 1-4 and yellow fever 17D vaccine strain, did not antagonize STAT5 phosphorylation. Mechanistically, WNV and ZIKV showed differential inhibition of Jak kinases upstream of STAT5, suggesting divergent countermeasures to inhibit STAT5 activation. Combined, these findings identify STAT5 as a target of antagonism by specific pathogenic flaviviruses to subvert antiviral immune responses in human DCs.

microbiology↗

bioRxiv2019

West Nile Virus Subverts T Cell Stimulatory Capacity of Human Dendritic Cells

West Nile virus (WNV) is a neurotropic flavivirus and the leading cause of mosquito-borne encephalitis in the United States. Recent studies in humans have found that dysfunctional T cell responses strongly correlate with development of severe WNV neuroinvasive disease. However, the contributions of human dendritic cells (DCs) in priming WNV-specific T cell immunity remains poorly understood. Here, we demonstrate that human monocyte-derived DCs (moDCs) support productive viral replication following infection with a pathogenic strain of WNV. Antiviral effector gene transcription was strongly induced during the log-phase viral growth, while secretion of type I interferons (IFN) occurred with delayed kinetics. Activation of RIG-I like receptor (RLR) or type I IFN signaling prior to log-phase viral growth significantly diminished viral replication, suggesting that activation of antiviral programs early can block WNV infection. In contrast to the induction of antiviral responses, WNV infection did not promote transcription or secretion of pro-inflammatory (IL-6, GM-CSF, CCL3, CCL5, CXCL9) or T cell modulatory cytokines (IL-4, IL-12, IL-15). There was also minimal induction of molecules associated with antigen presentation and T cell priming, including the co-stimulatory molecules CD80, CD86, and CD40. Functionally, WNV-infected moDCs dampened allogenic CD4 and CD8 T cell activation and proliferation. Combined, we propose a model where WNV subverts human DC activation to compromise priming of WNV-specific T cell immunity.\n\nImportanceWest Nile virus (WNV) is an encephalitic flavivirus that remains endemic in the United States. Previous studies have found dysfunctional T cell responses correlate to severe disease outcomes during human WNV infection. Here, we sought to better understand the ability of WNV to program human dendritic cells (DCs) to prime WNV-specific T cell responses. While productive infection of monocyte-derived DCs activated antiviral and type I interferon responses, molecules associated with inflammation and programming of T cells were minimally induced. Functionally, WNV-infected DCs dampened T cell activation and proliferation during an allogeneic response. Combined, our data supports a model where WNV infection of human DCs compromises WNV-specific T cell immunity.

microbiology↗

bioRxiv2019

Proteomic profiling of the oncogenic septin 9 reveals isoform-specific interactions in breast cancer cells

Septins are a family of multimeric GTP-binding proteins, which are abnormally expressed in cancer. Septin 9 (SEPT9) is an essential and ubiquitously expressed septin with multiple isoforms, which have differential expression patterns and effects in breast cancer cells. It is unknown, however, if SEPT9 isoforms associate with different molecular networks and functions. Here, we performed a proteomic screen in MCF-7 breast cancer cells to identify the interactome of GFP-SEPT9 isoforms 1, 4 and 5, which vary significantly in their N-terminal extensions. While all three isoforms associated with SEPT2 and SEPT7, the truncated SEPT9_i4 and SEPT9_i5 interacted with septins of the SEPT6 group more promiscuously than SEPT9_i1, which bound predominately SEPT8. Spatial mapping and functional clustering of non-septin partners showed isoform-specific differences in interactions with proteins of distinct subcellular organelles (e.g., nuclei, centrosomes, cilia) and functions such as cell signaling and ubiquitination. Notably, the interactome of the full length SEPT9_i1 was more enriched in cytoskeletal regulators, while the truncated SEPT9_i4 and SEPT9_i5 exhibited preferential and isoform-specific interactions with nuclear, signaling and ubiquitinating proteins. These data provide evidence for isoform-specific interactions, which arise from truncations in the N-terminal extensions of SEPT9, and point to novel roles in the pathogenesis of breast cancer.

cell biology↗