bluuebunny/crossref_metadata_2025 huggingface dataset:
Imaging of lung resident memory CD8+ T cells reveals antigen-specific early protective responses during secondary infection
Authors: van de Wall Stephanie , Anthony Scott M , Varga Steven M , Langlois Ryan A , Zehn Dietmar , Badovinac Vladimir P , Harty John T
Published: August 2023

Tissue-resident memory T (TRM) cells are poised at mucosal sites to confer robust protection against secondary infection. In contrast to skin and intestine, lung TRM cells are short lived with a decline in number coinciding with a decline in protective immunity against subsequent heterosubtypic influenza infections. Despite this causal relationship, the localization, dynamics and function of lung Trm during secondary responses remains poorly defined. To address this, we used two-photon intravital microscopy of lungs to assess in real time the spatiotemporal dynamics of lung TRM cells during recall infection. At a memory time point, lung TRM cells (representing both CD103+ and CD103− subsets) exhibit significantly lower motility and less displacement compared to an CD8+ effector T cells at an earlier time point post infection. Depletion of circulating CD8+ T cells using a low dose antibody administered systemically led to enrichment of lung TRM cells that exhibited slow to moderate speed scanning patterns. A fate mapping model for CD103+ TRM cells specifically revealed limited speed (<5 um/min) and patrolling behavior compared to CD103− TRM cells. Within 24 hr of rechallenge, CD103+ TRM cells co-localized with infected cells and by 48 hr, lung-resident CD8+ T cells exhibit significantly lower motility with recruitment and clustering in infected regions in an antigen-specific manner. Protective immune responses by lung TRM cells is further accompanied by CXCR3-dependent migration of dendritic cells to draining lymph nodes as well as recruitment of inflammatory monocytes. Overall, these data unravel the spatiotemporal dynamics of lung TRM cells and interactions with recruited immune cells for early protective immunity.

Supported by grants from NIH (GM134880, AI42767, AI114543, AI151183, AI167249, the Holden Cancer Center at the University of Iowa and its National Cancer Institute Award P30CA086862
Double negative T cell-NK cell axis orchestrates antitumor immunity against triple negative breast cancer
Authors: Zhang Hui , Little Alex A
Published: August 2023

Triple-negative breast cancer (TNBC) is hard to treat because of the development of chemoresistance and metastasis. It is recently found that the combination of chemotherapy and immune checkpoint blockade can significantly improve TNBC treatment. However, the low response rate to immunotherapy greatly hampers the application of immunotherapy to TNBC. It is urgent to find new paradigms to improve immunotherapy for TNBC. We previously found that transferring thymocytes into Ja281 KO mice could inhibit E0771 TNBC formation. Herein, we further determine the underlying mechanism. We found that transferring splenocytes, CD4+, or CD8+ T cells into the Ja281 KO mice could not induce antitumor immunity. Transferring thymic double-negative T (DN T) cells into the Ja281 KO mice could inhibit tumor formation. Treating the cell-transferred Ja281 mice with anti-TCRβ or anti-TCRγδ antibodies two days before tumor inoculation, E0771 TNBC grew in the anti-TCRβ treated mice but not anti-TCRγδ-treated mice. These results indicated that the cell transfer-induced antitumor immunity in Ja281 KO mice was mediated by TCRαβ+ DN T cells. Transferring iNKT or MAIT KO DN T cells did not affect cell-transfer-induced antitumor immunity. Only NK cell depletion abrogated DN T transfer-mediated antitumor immunity by using an antibody to deplete NK or macrophage, suggesting that NK cells are essential for DN T-mediated antitumor immunity against E0771. Antibody depletion of the circulating transferred cells did not affect cell transfer-induced antitumor immunity, suggesting that the DN T cells mediating antitumor immunity are tissue-resident cells. Collectively, these data indicate that the DN T/NK cell axis orchestrates antitumor immunity against TNBC.

Supported by Washington State University College of Pharmacy and Pharmaceutical Sciences Start-up funds
Tumor infiltrating natural killer cells in soft tissue sarcoma are predominantly CD56dim: Implications for outcomes
Authors: Sholevar Cyrus , Cruz Sylvia M , Iranpur Khurshid , Judge Sean J , Gingrich Alicia , Farley Lauren E , Razmara Aryana , Lammers Marshall , Thorpe Steven , Monjazeb Arta M , Murphy William J , Canter Robert J
Published: August 2023

Natural killer (NK) cells have been shown to be important mediators of anti-tumor responses, including in soft tissue sarcomas (STS). NK cells show significant heterogeneity, depending on maturation, tissue residency, and inflammatory environment. As previous studies have suggested that tumor-infiltrating NK cells (TiNKs) acquire a less cytotoxic CD56bright tissue resident phenotype, we sought to compare blood versus tumor NK cell phenotype in STS and evaluate any association with survival. Blood and tumor from 27 STS patients undergoing surgery from 2018–2022 were collected prospectively for flow cytometry and retrospectively analyzed. 52% were female, the mean age was 59, and 74% were AJCC stage 3. Increasing absolute number of NK cells in the blood correlated with longer survival (P<0.005, r=0.6). As expected, CD56dim NK cells predominated in the blood (91.5±5.8% of CD3-CD56+ lymphocytes compared to 80.1±13% in tumor, P<0.005). In contrast, CD56bright cells were enriched in tumor representing 18.5±13% compared to 8.4±5.8% in blood (P<0.005). Although CD56bright NK cells were approximately 2.4±3-fold higher in tumor compared to blood, CD56dim NK cells still represented the majority of TiNKs. Higher proportions of both overall NK cells and CD56dim NK cells in STS tumors were associated with better metastasis-free survival on Kaplan-Meier analysis (P<0.05). CD56dim TiNKs had higher NKp46 expression than CD56brights. In conclusion, both blood and intra-tumoral NK cells appear prognostic in STS. Although the proportion of CD56bright TiNKs in STS is higher than blood the majority of TiNKs are CD56dim consistent with a cytotoxic phenotype. Better characterization of NK subsets in STS is likely to have translational significance.
Identifiation of novel effector regulatory T cells through simulaneous single cell epigenetic and transcriptomic profiling
Authors: Nam Hyo Jeong , Kim Yong-Hee , Lee Woochan , Byun Nari , Yoon Jeong Heon , Kim Yong Chan , Chung Hyunwoo , Jeong Soyoung , Joh Christine , Kang Sung-Jun , Park Kyunghyuk , Cha Bukyoung , Kim Jong-Il , Park Chung-Gyu , Kim Hyun Je
Published: August 2023

Regulatory T cells (Tregs) harness a central role in peripheral immune tolerance. Since its discovery, there have been numerous efforts to dissect the role of Tregs in the context of disease condition. Recently, Treg fields are moving from the academia to industry since many evidences have been accumulated for the potential of treatment of inflammatory disease by administering genetically engineered Tregs. However, in the pre-clinical or clinical trials, this rare cell subset needs to be expanded in ex-vivo condition to achieve a sufficient number for the controlling of the disease. Our group previously established ex-vivo Treg expansion condition with 25 mer oligonucleotide to maintain the stability and function of Tregs. However, non-biased investigations on the perturbation of transcriptomic and epigenetic changes during the culture condition has not been explored. Since cutting edge multiomics tools which can simultaneously profile epigenetic and transcript signatures in a single cell resolution has been commercialized, we adopt 10x multiome technique to investigate our frozen ex-vivo expanded Tregs(eTreg) compared to freshly isolated frozen human Treg(fTreg) from the same donor. Compared to fTreg cells, we have identified a new subset that exists only in the eTreg cells and named it eeTreg. We found that the eeTreg resembles the phenotype of both effector Tregs and tumor-infiltrated Tregs. eeTreg is characterized by a significantly increased expression and chromatin accessibility score of FOXP3, STAT5B, ETS1 and RUNX1, which are known to be the essential transcription factors of regulatory T cell function. In summary, we suggest a novel human treg subset which might be manufactured by manipulating ex-vivo culture condition.
PD1 blockade improves survival during lethal microbial exposure in old mice
Authors: Dahlquist Korbyn J V , Huggins Matthew A , Yousefzadeh Matthew J , Soto-Palma Carolina , Cholensky Stephanie H , Pierson Mark , Hamilton Sara E , Camell Christina D
Published: August 2023

The elderly and those with chronic diseases have a 2 to 10-fold increased risk of hospitalization and mortality following infection as compared with healthy adults. A significant contributor to disease severity is the aging of the immune system, including an expansion of exhausted T cells. These cells simultaneously promote excessive levels of inflammation and display impaired T cell activation and function. How exhausted T cells from aged individuals respond to infection and whether acute restoration of T cell activation further exacerbates the inflammatory environment or generates functional T cells is unclear. Previously, we used a mouse model of normal microbial exposure (NME), where old specific pathogen-free (SPF) mice show 100% mortality following exposure to multiple microbes ordinarily found in pet store mice. In the present study, we show that when exposed to NME, old SPF mice have increased expression of multiple inflammatory pathways and exhibit elevated frequencies of a heterogenous, exhausted CD8+ T cell pool that expresses differing combinations of programmed cell death protein 1 (PD1), TOX, and CXCR5. Pre-treatment or intervention with an anti-PD1 monoclonal blocking antibody during the exposure of old SPF mice to NME significantly improves both antibody production and survival, without altering the acute inflammatory response. Anti-PD1 checkpoint blockade-mediated survival is dependent on CD8+, but not CD4+ T cells. Correspondingly, CD8+ PD1+ T cells from old mice given anti-PD1 monoclonal antibody have increased granzyme B production. These data reveal a new approach for reducing vulnerability to infections in the elderly by targeting CD8+ T cell exhaustion through PD1 checkpoint blockade immunotherapy.

This work was supported by National Institute on Aging grant R00 AG058800 (C.D.C.), R21AG078638, the Fesler-Lampert Chair in Aging Studies, the Glenn Foundation for Medical Research/AFAR Grants for Junior Faculty, Irene Diamond Fund/AFAR Postdoctoral Transition Award (M.J.Y) and the Medical Discovery Team on the Biology of Aging.
PD-1 protects expanding human T cells from premature restimulation-induced cell death
Authors: Lee Katherine , Elster Sara , Lake Camille M , Snow Andrew L
Published: August 2023

Programmed cell death-1 (PD-1) is a co-inhibitory protein that dampens TCR/CD28 signaling in part by recruiting the phosphatase SHP2 to the immunological synapse. Chronic antigenic restimulation of T cells can induce apoptosis or elicit PD-1-mediated exhaustion, rendering them hyporesponsive and ineffectual. While the checkpoint function of PD-1 has profound implications for cancer immunotherapy, the role of PD-1 in conventional effector T cell responses remains unclear. Restimulation-induced cell death (RICD) is a critical propriocidal apoptosis program triggered in activated T cells upon robust TCR re-engagement, constraining effector T cell expansion and longevity to prevent collateral tissue damage. We hypothesized that PD-1 attenuates RICD sensitivity in human effector T cells by modulating TCR signal strength. Here we show that transient upregulation of PD-1 helps to protect newly activated human CD4+ and CD8+ T cells from premature RICD during clonal expansion, with lesser effects noted in terminally differentiated T cells. Both antibody-mediated blockade of PD-1-PD-L1 and SHP2 inhibition increased RICD sensitivity to a similar extent. Conversely, concomitant addition of recombinant PD-L1 with TCR restimulation significantly reduced RICD in a dose-dependent manner, with more pronounced protection noted uopn CD28 coligation. Our ongoing work aims to define the role of recently discovered downstream PD-1 signaling nodes (e.g. PAG, VRK2) and specific pro/anti-apoptotic proteins in this process. These findings present significant implications for understanding how PD-1 blockade may influence apoptosis sensitivity in both normal and exhausted T cells to alter adaptive immune responses.

Supported by grants from NIH (R35GM139619)
Ets1 represses a T-bet dependent migration program and stabilizes the iNKT1 adhesome
Authors: Morgan Roxroy C , Bartom Elizabeth T , Sigvardsson Mikael , Maienschein-Cline Mark , Kee Barbara L
Published: August 2023

iNKT cells are innate-like T cells that recognize glycolipids presented on MHCI-like CD1d using a semi-invariant TCR. Upon activation, iNKTs rapidly produce cytokines to direct the adaptive response towards a TH1, TH2 or TH17 response. The transcription factor ETS1 is important for iNKT cell development, but its roles in iNKT function and homeostasis are not well characterized. Utilizing a novel mouse model for targeted deletion of Ets1 in iNKT1s, we show that ETS1 limits expression of T-bet, the signature transcription factor of the iNKT1 lineage. Ets1Δ/Δ hepatic iNKT1 cells upregulated a gene program associated with migration and had reduced expression of LFA-1 and ICAM-1, receptors critical for retention in the liver. Remarkably, Ets1Δ/Δ liver iNKT1s remained tissue resident but no longer required LFA-1/ICAM-1 for retention. Heterozygous deletion of T-bet abrogated expression of multiple migration-associated genes, restored LFA-1 and ICAM-1, and re-established reliance on LFA-1/ICAM-1 for retention. Our data demonstrate that ETS1 controls the mechanisms of hepatic iNKT1 cell residency by limiting a T-bet-dependent migration and adhesion program.

Supported by NIH/NIAID grant R01 AI106352
Characterizing the Immune Cell Transcriptomic Response To Low-Dose Ionizing Radiation
Authors: Marr Bryan , Khan Abrar , Blimkie Melinda , Pashangzadeh Salar , Azzam Edward , Laakso Holly , Lee Seung-Hwan
Published: August 2023

Low-dose exposure to ionizing radiation is increasingly common in medical, industrial, and public settings worldwide. Previous studies examining the impacts of low-dose radiation (LDR) exposure on genetic material, cellular responses, and health have been inconclusive and conflicting. The National Academy of Science, Engineering and Medicine has outlined that future LDR research should utilize single-cell omics technologies and computational workflows to define the molecular signatures of LDR exposure. In partnership with the Canadian Nuclear Laboratories (CNL), we have used single-cell RNA sequencing (scRNA-seq) to characterize the in-vivo immune cell response to chronic LDR. Mature adult (18 weeks-old) C57BL/6 female mice were whole-body-exposed to 60Co gamma radiation for 7 days, at dose rates of 0.06 mGy/h or 0.6 mGy/h, to achieve cumulative absorbed doses of 10 mGy (n=6) or 100 mGy (n=6), respectively. scRNA-seq of cells isolated immediately post-irradiation identified subtle candidate transcriptomic changes caused by LDR exposure in splenic and bone marrow cell populations. As the first scRNA-seq study of in-vivo LDR exposure, we reveal a more detailed description of the cellular response to LDR exposure than available in the current literature. Therefore, dissemination of these results will advance radiation biology and will be valuable for evaluating Nuclear Safety guidelines.

The research is funded by the CANDU Owner’s Group and the Mitacs Accelerate program.
Hypoxia promotes cytolytic activity of CD8 T cells and pathogenesis in cutaneous leishmaniasis
Authors: Fowler Erin , Amorim Camila Farias , Hales Emily D S , Varkey Aditi , Salem Mariam , Xin Gang , Collins Patrick L , Novais Fernanda
Published: August 2023

Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania and the most common form of the disease is cutaneous leishmaniasis (CL). A fundamental question in CL is what regulates the development of severe disease, which is critical to develop therapies to ameliorate pathology. In a series of studies, we demonstrated CD8 T cell-dependent cytotoxicity as the main inducer of immunopathology in CL. This result was unexpected since IFN-γ production by CD8 T cells plays a protective role by promoting pathogen elimination. To resolve this paradox, we studied the CD8 T cells in different anatomic sites and found that the effector function of CD8 T cells in CL depends on their location: while CD8 T cells are cytotoxic (GzmB+) and produce little IFN-γ in the skin lesions, CD8 T cells in the draining lymph nodes (dLN) have the opposite profile. Importantly, GzmB-CD8 T cells from dLN quickly upregulate GzmB after injection into CL lesions. By transcriptional profiling, we found that CD8 T cells in lesions but not dLN have a hypoxic signature. In vivo, we observed that CL lesions are hypoxic using the Oxyphor G4 oxygen probe and pimonidazole staining. In vitro, we found that induction of hypoxia was sufficient to convert GzmB- into GzmB+ CD8 T cells, and significantly decreased CD8 T cell production of IFN-γ. In vivo, blocking hypoxia-inducible factor (HIF), a master regulator of hypoxia, with acriflavine decreased lesion development in mice. Mice with CD8 T cell deficient in HIF-1α produced significantly less GzmB in the skin lesions compared to wildtype mice. Together, our results suggest that the hypoxic signaling through the transcription factor HIF in CL lesions is necessary to convert CD8 T cells into pathogenic CD8 T cells in the skin lesion.

Supported by grants from NIH (1R01AI162711)
Protective Role of IL-17 in Pulmonary Tuberculosis Relapse
Authors: Clark Lucinda Puebla , Martinez-Martinez Yazmin Berenice , Huante Matthew B , Chauhan Sadhana , Files Megan A , Endsley Janice J
Published: August 2023

Tuberculosis (TB) recurrence following drug treatment and apparent clinical cure affects an estimated 700,000 people annually and is increasingly associated with relapse of initial Mycobacterium tuberculosis (Mtb) infection. Cellular immunity by T helper (Th) cells, including Th17 populations is important during active TB. The contribution of Th17 and other Th populations to immune pressure and Mtb clearance following TB chemotherapy is poorly understood. We used Mtb-HN878 for a paucibacillary model of TB infection in WT, IL17RA-KO, and second study using WT and conditional Th17-KO (CD4-Cre-RORγtflox/flox) mice to identify the cellular sources and protective role of IL-17 in TB relapse following drug therapy. Assessment of disrupted lung by multi-parameter flow cytometry demonstrated that Th17 populations expand during active TB, retract after drug therapy, and subsequently display a moderate re-expansion during relapse. Flow cytometric analysis also revealed expansion and contraction of other IL-17+ cells among both the non-lymphocyte and CD3− lymphocyte populations in the lung during active, post-drug, and relapse phase of TB. Compared to WT, the lung of IL-17RA-KO and conditional Th17-KO mice had greater mycobacterial burden during TB relapse. These results demonstrate a critical role for Th17 cells and IL-17 signaling in protection against Mtb regrowth following TB chemotherapy. Further, it suggests that both lymphoid and non-lymphoid cells of the innate immune system also contribute to IL-17-mediated protection during active and relapse TB.

NHI grant R01AI147948
TcpC affects the grazing resistance of Escherichia coli to the social amoeba Dictyostelium discoideum
Authors: Hamilton Sarah , Akingbehin Victoria , Palm Cheyenne , Snyder Michelle
Published: August 2023

Uropathogenic Escherichia coli (UPEC) is among the most common causative agents of urinary tract infections (UTIs). UPECs survive in hosts using various mechanisms, including by secreting a protein known as TcpC, which is a Toll/interleukin-1 receptor (TIR) domain containing protein. In mammals, TIR domains are important components of Toll like receptor (TLR) signaling pathways that activate innate immune responses. TcpC interacts with components of TLR signaling, inhibiting activation of the immune system and preventing the detection and destruction of the bacteria in mammalian hosts. We hypothesize that TcpC also affects the survival of UPECs in model systems, including the social amoeba Dictyostelium discoideum and planaria flatworms. To test this hypothesis, we are using mutant strains of the UPEC CFTO73 that have altered expression of TcpC to analyze the effects of TcpC on bacterial growth and virulence in model organisms. Our initial tests with D. discoideum indicate that the presence of TcpC in UPEC strains may increase their resistance to grazing and phagocytosis by the social amoeba D. discoideum. If TcpC affects survival of UPECs in model systems such as D. discoideum, this may open new avenues for characterization of the molecular mechanisms used by bacterial TIR domains to evade innate immune cells.

VA was supported by NSF LSAMP Award #1619676 and CP was supported by the NIH Bridges to Doctorate Scholars Program, NIH- R25 GM119970.
Antibiograms of select anaerobic bacteria over 10 years
Authors: Sterner Rosalie , Shannon Samantha , Estes Lynn , Norgan Andrew , Schuetz Audrey
Published: August 2023

Antibiotics are critical complements to the immune response in combatting infection. Anaerobic bacteria are increasingly common causes of infection, and antibiotic resistance of anaerobes has been rising. Most laboratories do not perform antimicrobial susceptibility testing of anaerobes, and even fewer have compiled antibiogram data. Here we investigated antimicrobial susceptibility results of anaerobes from January 2012 to December 2021 obtained from inpatients at Mayo Clinic using reference agar dilution testing. Actinomyces (60–70 isolates annually), Bacteroides fragilis (30–80), Finegoldia magna (40–60), and Cutibacterium acnes (70–110) were included. Penicillin, clindamycin, metronidazole, ertapenem, piperacillin-tazobactam, and moxifloxacin were tested. Clinical and Laboratory Standards (CLSI) breakpoints were followed. B. fragilis showed ≥90% and Actinomyces 100% susceptibility to piperacillin-tazobactam and ertapenem. All F. magna and >95% B. fragilis were susceptible to metronidazole. The most striking fluctuations in percent susceptibility from year to year were observed for clindamycin, with Actinomyces ranging from 66–79%, B. fragilis 34–67%, and F. magna 47–85%. In the absence of local anaerobe antibiograms, providers may refer to such antibiogram data to guide empiric anti-anaerobic therapy at their institutions. In conclusion, anaerobe antibiotic susceptibility patterns must be monitored to ensure appropriate antibiotic empiric therapy and stewardship.
Mathematical modeling suggests heterogeneous replication of Mycobacterium tuberculosis in rabbits
Authors: Ganusov Vitaly V , Kolloli Afsal , Subbian Selva
Published: August 2023

Tuberculosis (TB), the disease caused by Mycobacterium tuberculosis (Mtb), remains the major health problem. We performed experiments with rabbits infected with a novel, virulent clinical Mtb isolate of the Beijing lineage HN878 carrying an unstable plasmid pBP10, allowing us to quantify how quickly HN878 replicates and dies in lungs of rabbits. In our in vitro experiments we found that pBP10 is more stable in HN878 strain than in a more commonly used laboratory-adapted H37Rv strain (the segregation coefficient being s=0.10 in HN878 vs. s=0.18 in H37Rv). Interestingly, the kinetics of the frequency of plasmid-bearing bacteria in lungs of Mtb-infected rabbits did not follow an expected monotonic decline; the percent of Mtb-bearing cells increased between 4 and 8 weeks post-infection and remained stable between 12 and 18 weeks post-infection despite a large increase in bacterial numbers in the lung at these time points. Mathematical modeling suggested that such a non-monotonic change in the percent of plasmid-bearing cells can be explained if the lung Mtb population consists of several (at least 2) sub-populations with different replication kinetics: one major population expanding early and being controlled, while another, a smaller population expanding at later times causing a counterintuitive increase in the percent of plasmid-bearing cells. Given that HN878 forms well circumscribed granulomas in rabbits, our results suggest independent bacterial dynamics in subsets of such granulomas. Our model predictions can be tested in experiments in which HN878-pBP10 dynamics in individual granulomas is followed over time.

1R01AI158963-01A1
Elucidating the role of the host protein, FBXO22, in the infection of macrophages with the zoonotic bacterial pathogen, Brucella
Authors: Mazumdar Varadendra Balaji , Joshi Kirnami , Nandi Binita Roy , Radhakrishnan Girish K
Published: August 2023

Brucella spp. are facultative intracellular bacterial pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes. Successful invasion and chronic intracellular persistence indicate that Brucella manipulates various host cellular processes to create a replication permissive niche. The essential mechanisms and host factors that support the invasion and intracellular replication of Brucella remain poorly understood. RNA interference (RNAi) is a potent molecular tool for understanding gene function by downregulating the target gene expression through mRNA degradation. We used siRNA-based screening to identify the host factors that are involved in Brucella-macrophage interaction. Macrophages were treated with gene specific siRNAs, followed by infection with B. neotomae/B. melitensis and quantification of intracellular Brucella. The screening identified several host factors that are involved in the Brucella infection of macrophages. Subsequently, we performed detailed characterization of one of the identified host proteins, FBXO22. Downregulation of FBXO22 resulted in diminished uptake of Brucella into macrophages, which was dependent on NF-κB-mediated regulation of phagocytic receptors. FBXO22 expression was upregulated in Brucella-infected macrophages that resulted in induction of phagocytic receptors and enhanced production of pro-inflammatory cytokines through NF-κB. Furthermore, we found that FBXO22 recruits the effector proteins of Brucella, including the anti-inflammatory proteins, for degradation through the SCF complex.

Funding from Department of Biotechnology (DBT) (BT/PR12301/ADV/90/176/2014)
Blimp-1 is a negative regulator of IL-2 signaling in T cells
Authors: Roy Suyasha , Ren Min , Christensen Steve , Li Peng , Spolski Rosanne , Phelan James , Staudt Louis , Leonard Warren J
Published: August 2023

Interleukin-2 (IL-2) is a T cell growth factor with immunomodulatory effects and pleiotropic actions on T cells. IL-2 signaling occurs via the intermediate IL-2Rb and IL-2Rg and high-affinity IL-2Ra receptors, fine-tuning tolerance and effector functions. To investigate key regulators of the IL-2 signaling pathway, we performed genome-wide CRISPR-Cas9 screening in IL-2-dependent ED40515(+) cells, which were derived from a patient with HTLV-I associated Adult T Cell Leukemia. We found a significant dropout of sgRNAs for genes known to be important in IL-2 signaling, whereas sgRNAs targeting Prdm1 were highly enriched, with its being the top enriched hit in the screen. Prdm1 encodes Blimp-1, which is known to inhibit IL-2 production in T cells; however, its precise role in IL-2 signaling pathway remains elusive. Overexpression of Prdm1 resulted in lower expression of IL-2R subunits (CD25, CD122, CD132) and decreased IL-2-induced phosphorylation of STAT5, AKT, ERK in mouse CD4+ T cells. Correspondingly, conditional knockout (CKO) of Prdm1 in mouse CD4+ T cells and Tregs led to enhanced expression of IL-2R subunits, pSTAT5, pAKT and pERK. In an influenza infection mouse model, CD122 expression and pSTAT5 levels were higher in T follicular helper (TFH) cells from Prdm1 CKO mice as compared to WT TFH cells. Further, adoptive transfer of Prdm1 CKO Tregs into Rag1−/− mice resulted in augmented IL-2 signaling and ameliorated inflammation in colitis. Concordantly, CRISPR/Cas9-mediated deletion of Prdm1 in human CD4+ T cells and Tregs resulted in increased expression of IL-2R subunits and IL-2-induced phosphorylation of STAT5, AKT and ERK. These results suggest that Blimp-1 is a negative regulator of IL-2 signaling in mouse and human T cells.

Supported by intramural funding from NIH
14-3-3zeta Regulates Transmembrane Receptor Signaling by Binding TRAF Proteins
Authors: Ayyasamy Rathinam , Chattopadhyay Saurabh , Chakravarti Ritu
Published: August 2023

Adaptor proteins are critical for signal transduction events involving cytokine signaling, host-response, and autoimmune diseases. Our laboratory is interested in understanding the immune regulatory role of 14-3-3zeta (z), an adaptor protein, in signal transductions and diseases. We examined the role of 14-3-3z in interleukin-17 (IL-17A) signaling and autoimmune inflammatory arthritis using genetically modified cells and rats. The IL-17A signal transduction requires IL-17 receptor cytoplasmic domain interactions with Act1 and several TRAF proteins. Our results show that 14-3-3z interacts with TRAF6, TRAF2, and TRAF5 upon IL-17A stimulation which has functional consequences. The interaction of 14-3-3z and TRAF protein was found to be specific, as mutation of the interacting residues resulted in the loss of binding with functional impacts. Further interaction studies show a higher affinity of 14-3-3z for TRAF6, which has two putative 14-3-3 binding sites. To examine the generality of 14-3-3z-TRAF interactions and their role in another transmembrane receptor signaling, we examined RANKL signal transduction. Similar to IL-17A, 14-3-3z-TRAF interaction was increased upon RANKL treatment and had functional consequences. While there are common domains and significant sequence conservation between the TRAF proteins, a specificity of interactions exists with 14-3-3z. Our results show that the 14-3-3z-TRAF axis is a novel regulatory mechanism underneath several transmembrane receptor-mediated signaling. Current studies target understanding the molecular basis of interactions between 14-3-3z and specific TRAF proteins.
Development of a specific, active human IL-18 assay for assessing inflammasome activation
Authors: Mamott Daniel B , Mahan Sarah , Cali Jim , Lazar Dan , O’Brien Martha
Published: August 2023

Interleukin-18 (IL-18) was first identified for its ability to induce interferon-γ expression. IL-18 is a proinflammatory cytokine and its release along with IL-1β is a primary output of inflammasome activation. We have developed convenient tools for monitoring inflammasome activation, including assays for caspase-1 activity and IL-1β release. While both IL-1β and IL-18 are expressed as inactive zymogens requiring processing to active forms, they differ in expression and regulation. IL-1β requires upregulation for expression and is primarily restricted to myeloid cells, whereas IL-18 is constitutively expressed more broadly in epithelial cells, as well as myeloid cells. IL-18 is also uniquely regulated by an IL-18 binding protein (IL-18bp). The IL-18bp has a very high affinity for active IL-18 and effectively blocks receptor activation. IL-18-stimulated interferon-γ release stimulates IL-18bp release in a negative feedback loop. The Lumit™ technology is a Nanoluc®-based platform that enables direct, no wash assays for analyte detection using standard luminometers. For Lumit IL-18 assays, we either NanoBiT®-labeled two IL-18 antibodies or NanoBiT®-labeled the IL-18bp as well as one IL-18 antibody. These resulted in, respectively, one assay that measures both IL-18 and proIL-18 and another assay specific for active IL-18. The specific assay was used to measure active IL-18 release from canonical inflammasome-activated THP-1 cells and PBMCs, as well as non-canonical inflammasome-activated lung A549 epithelial cells. Enabling direct quantitation of unbound, active IL-18 is essential for elucidating IL-18’s role in inflammation and disease. A Lumit™ IL-18 assay adds to the toolbox for monitoring inflammasome activation.
TRAF3 regulates IL-6 receptor signaling in T cells through the phosphatase PTPN22
Authors: Hornick Emma , Shao Peng , Butler Noah , Bishop Gail A
Published: August 2023

IL-6 is an important signal three cytokine for T helper 17 (Th17) and T follicular helper (Tfh) differentiation. Previous work has suggested a possible role for TRAF3 in the function and/or differentiation of these two subsets, but no direct studies have yet been performed. In this study, we examine the role of TRAF3 in IL-6R signaling in T cells. TRAF3 regulates IL-2R and type I interferon receptor signaling in T cells by altering recruitment of the phosphatases PTPN2 and PTPN22, respectively. B cell TRAF3 curtails IL-6R signaling by recruiting PTPN22, thus we hypothesized that TRAF3 regulates T cell IL-6R signaling through a PTPN22-dependent mechanism.

We investigated the role of T cell TRAF3 using a TRAF3-deficient human T cell lymphoma line and a T cells from a T cell-specific TRAF3 knockout mouse (T-Traf3−/−). We found reduced IL-6-induced STAT1 and STAT3 activation in human and mouse T cells. STAT1 and STAT3 activation are both required for full differentiation of mouse Tfh cells, whereas STAT3 is required for and STAT1 antagonizes Th17 differentiation. Interestingly, in vitro differentiation of naïve CD4 T cells to Th17 cells was not affected by TRAF3 deficiency. To determine the mechanism by which TRAF3 promotes STAT1 and STAT3 activation in response to IL-6R signaling, we treated WT and Traf3−/− T cells with IL-6 in the presence of PTPN22 inhibitor or vehicle control. We found that PTPN22 inhibition restored activation of STAT3, but did not restore activation of STAT1, suggesting different mechanisms of regulation. Together these data implicate TRAF3 in regulation of IL-6R signaling in a partly PTPN22-dependent manner, lending an additional level of understanding to TRAF3 regulation of Th17 and Tfh differentiation.

Supported by NIH R01 AI123107, R01 AI162656, T32 AI007260
B-cell activation state determines aryl hydrocarbon receptor regulation of CCR9 expression
Authors: Bauerle Logan V , Dekrey Gregory K
Published: August 2023

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates CCR9 expression on activated mouse B cells. Because AhR expression levels are themselves regulated by B cell activation state, in this study, we hypothesized that CCR9 regulation by the AhR would vary over time after B cell activation. This was tested in vitro using B cells isolated from mouse spleens (magnetic separation) followed by stimulation with 25 μg/mL LPS, 300 ng/mL anti IgD-dextran, and 2 ng/mL human TGF-Beta for 96 hours. Cells were exposed to AhR ligands at zero or 24 hours of culture as follows: 100 μM of the antagonist CH22319 or 50 nM of the agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). CCR9 expression was determined by flow cytometry. As was shown previously, AhR agonism at zero hours of culture significantly enhanced CCR9 expression, relative to AhR antagonism (P < 0.05). In contrast, AhR agonism at 24 hours of culture did not lead to significant alteration of CCR9 expression, relative to AhR antagonism. This finding suggests that migration of B cells to the gut may be enhanced by AhR activation, but only if AhR activation occurs simultaneously with antigen-mediated cell activation.

Supported by internal grants from the University of Northern Colorado, College of Natural and Health Sciences (NHS Student Research Fund, SBS Summer Research Fund).
Attenuated Salmonella blocks cancer metastasis by activating NK cells in an IFN-γ-dependent manner
Authors: Huang Jiandong , Lin Qiubin , Rong Li , Lin Shu-hai , Fu Guo
Published: August 2023

Metastasis accounts for 90% of cancer related deaths and blocking of metastatic cascade has critical clinical impact. However, the clinical drug development for cancer treatment, including cancer immunotherapies, is evaluated largely depending on their ability to cause tumour shrinkage and ignores the effect on metastasis as it has proven challenging to target. Therefore, there is an urgent need for novel therapeutic strategies and agents targeting metastasis. Using an attenuated Salmonella typhimurium strain YB1 engineered by our lab, we have found a potent suppressive effect of attenuated salmonella on cancer metastasis, regardless of cancer types and genetic background, by evoking strong anti-metastatic immune response. Further study showed that suppression of cancer metastasis by attenuated Salmonella only requires the innate immune response. Among the many induced cytokines, we identified IFN-γ as an indispensable factor for inhibiting cancer metastasis. Based on CyTOF (mass cytometry or cytometry by time of flight) analysis of the innate immune responses after Salmonella treatment and antibody-mediated cell depletion, we further demonstrated that NK cells are the major factor involved in Salmonella-provoked metastasis suppression. We found that IFN-γ was mainly produced by NK cells during early Salmonella infection, and in turn, IFN-γ promoted the accumulation, activation and cytotoxicity of NK cells. The IFN-γ-dependent NK cells directly eliminated newly accumulated cancer cells in the lung to block the cancer metastasis cascade in response to the Salmonella treatment.

This work was supported by Shenzhen Science and Technology Innovation Committee Basic Science Research Grant (JCYJ20170413154523577) to Dr. Jian-Dong Huang.
Targeting Fibrosis to Improve Cancer Outcomes
Authors: Breckenridge Joey Emery , Dell Garrison , Steele Hope , Cheng Jason Liang , Ghastine Andrew , Culberson Erica , Herro Rana
Published: August 2023

Scleroderma (SSc) is an idiopathic autoimmune connective tissue disease. The three hallmarks of SSc include 1) vasculopathy resulting in 2) progressive fibrosis or thickening of the skin, lungs, and internal organs and 3) aberrant immune infiltration. Fibrotic pulmonary involvement is the leading cause of death in SSc patients. Recent data from John Hopkins Scleroderma Center have shown a significantly increased risk of lung cancer in SSc patients. However, the mechanisms linking the two diseases are not well understood. Moreover, due to the close temporal relationship between SSc and lung cancer diagnosis, it is unclear whether lung fibrosis precedes tumorigenesis or conversely lung tumorigenesis precedes fibrosis. It has become evident that certain fibrotic diseases can progress towards cancer. Seemingly, non-alcoholic steatohepatitis or HBV/HCV infection-induced liver cirrhosis can progress to hepatocellular carcinoma. Vice versa, many solid tumors are fibrotic, and desmoplasia is referred to the pathological remodeling observed in the tumor microenvironment. There seems to be a tight link between fibrosis and cancer although the directionality of this progression is unknown. Moreover, several fibrotic processes involving the ß-catenin/Wnt, YAP/TAZ and hedgehog pathways in addition to fibrogenic growth factors (TGFß, VEGF, FGF) have been known to drive cancer. We are developing a murine model to study the mechanisms driving fibrosis to cancer progression in the lungs.
Proteolysis-targeting chimera against NR4A1 for cancer immunotherapy
Authors: Wang Lei , Xiao Yufeng , Master Rohan , Luo Yuewan , Zheng Guangrong , Zhou Daohong , Zhang Weizhou
Published: August 2023

Modulating tumor-infiltrating immune cells is the key mission for the development of cancer immunotherapy. Based on recent literature and our bioinformatic analysis, nuclear receptor subfamily 4 group A member 1 (NR4A1) is an ideal target for cancer immunotherapy due to its important role in T cell exhaustion, regulatory T (Treg) cell function. Here we targets NR4A1 via proteolysis-targeting chimeric (PROTAC) technology, expecting to achieve immune activation and cancer clearance.

We designed and synthesized over 80 NR4A1 PROTACs based on several reported NR4A1 ligands, such as celastrol. We found that several celastrol-based PROTACs achieved effective degradation of target protein NR4A1. We studied the therapeutic efficiency of the lead NR-V04, a celastrol- and von Hippel-Lindau (VHL)-based PROTAC. As expected, we confirmed that the NR-V04-induced NR4A1 degradation is through the uniquitin proteasome system with a tenery complex formation among NR4A1, VHL and NR-V04. The lead NR-V04 exhibits outstanding pharmacokinetics and effectively inhibits tumor growth at a low dose, 1.8mg/kg twice a week, in B16F10, Yummer 1.7, and MC38 mouse tumor models. We proved that NR-V04 significantly enhances anti-tumor immunity with seemingly distinct mechanisms of immune activation in a tumor-dependent manner. For example, NR-V04 induces the expansion of B cells and the reduction in myeloid-derived suppressor cells (MDSC) in B16F10; the reduction of Tregs, and the induction of CD8 effector population in Yummer 1.7 and MC38 tumors. we did not find obvious toxicity at these effective doses for up to 2 months. Therefore, the lead NR-V04 or other better ones under test have the potential for translation as a novel immunotherapeutic strategy.

BC200100 - “Developing a Novel PROTAC-Based NR4A1 Degrader for Breast Cancer Therapy” (DEPARTMENT OF THE ARMY)
YAP inhibition by CO promotes ferroptosis via ATF4-dependent REDD1 increase in melanoma
Authors: Park Jeongmin , Gong Jeong Heon , Joe Yeonsoo , Chung Hun Taeg
Published: August 2023

Ferroptotic cell death is a recently recognized form of non-apoptotic regulated cell death and exerts cytotoxicity in cancer cells. Carbon monoxide (CO) as an endogenous physiological mediator confers therapeutic potential in various cancer, while the therapeutic effects of CO on melanoma and its mechanism remain elusive. In this study, we suggest that CO induces ferroptosis via YAP inhibition and ATF4 activation in melanoma. YAP phosphorylation on serine 127 by CO-releasing molecule 2 (CORM2) caused cytoplasmic retention of YAP in B16F10 mouse melanoma cells, resulting in inhibition of YAP activity for transcription of target genes. Thus, CO-mediated YAP inhibition decreased SLC7A11 expression in melanoma cell lines, leading to ferroptosis. Moreover, CO increased REDD1 expression through PERK-ROS-ATF4 pathway. ATF4-REDD expression increased by CO became vulnerable to ferroptosis in melanoma. Thus, CO-induced YAP inhibition can induce ferroptosis via ATF4-mediated REDD1 increase and inhibition of SLC7A11. Furthermore, CO gas inhalation significantly inhibited B16F10 tumor size in xenograft mouse models resulting from ferroptosis induction via down-regulation of SLC7A11 and up-regulation of REDD1. These results suggest that CO acts as a potent inducer of ferroptosis in melanoma and that CO critically depends on YAP inhibition and ATF4 activation to promote ferroptosis.
Advancing Immunovirotherapy with Zika virus to treat high-grade brain tumors
Authors: Nair Sharmila , Chheda Milan , Diamond Michael S
Published: August 2023

High-grade brain tumors such as glioblastoma multiforme (GBM) are the most aggressive primary brain tumor and virtually all patients die within 2 years of diagnosis. Poor patient outcomes are caused by two known factors. First, presence of GBM stem cells (GSCs) which are a subpopulation of tumor cells that are resistant to radiation treatment and chemotherapy. GSCs drive tumor initiation and are the primary cause for tumor relapses. Currently, no treatment consistently kills these highly resistant cells. Second, the cold tumor microenvironment is filled with anti-inflammatory T cells and myeloid suppressor cells contributing to a weak anti-tumor immunological response. Oncolytic viral treatments are considered an attractive treatment option for high-grade solid tumors as they are engineered to replicate and destroy transformed cells as well as improve inflammatory immune responses against the tumor. However, none are specifically designed to kill GSCs. We previously tested and demonstrated that the natural tropism of Zika virus (ZIKV) can be harnessed to target and kill GSCs.

In vivo we demonstrated that intratumoral ZIKV treatment was enough to improve the survival outcomes of glioma bearing mice up to 40–60%. Importantly the oncolytic action of ZIKV extended beyond its anti-GSC effects. Reduction in tumor size and improved survival outcomes following oncolytic ZIKV treatment was caused by Perforin 1 activity in CD8+ T cells which clears tumor components not directly killed by ZIKV. In preliminary studies we observed that DC-2 and CCR2+ antigen presenting cell (APC) subsets are utilized by ZIKV to drive CD8+ T cells against GBM. This highlights the immunological barriers of GBM that can be overcome with ZIKV immunovirotherapy.
β-catenin inhibition using XAV939 nanoparticle promotes immunogenic cell death and suppresses tumor progression
Authors: Antony Ferrin , Kang Xeujia , Chen Pengyu , Babu Jayachandra , Suryawanshi Amol
Published: August 2023

Although the incidence of conjunctival melanoma is rare, it is a highly metastatic tumor with high recurrence and mortality rates. The current conjunctival melanoma treatment strategies, including surgical resection combined with chemo- or cryotherapy, have limited usefulness because of systemic side effects, recurrence, and metastases. Tumor-intrinsic Wnt/β-catenin pathway mediates immune exclusion in the tumor microenvironment (TME) and promotes tumorigenesis. Previous studies have shown that XAV939, a potent tankyrase inhibitor, can be used for the pharmacological inhibition of β-catenin. In this study, using tocopheryl polyethylene glycol 1000 succinate (TPGS)-based nanoparticle formulation for XAV939, we show that β-catenin inhibition suppresses melanoma cell proliferation and migration. Additionally, XAV939 nanoparticles promoted immunogenic cell death (ICD) of tumor cells with a significant extracellular release of ICD molecules, including HMGB1, calreticulin, and ATP. Further, using a mouse model of conjunctival melanoma, we show that β-catenin inhibition significantly suppresses conjunctival melanoma progression. Collectively, the local delivery of β-catenin represents a novel and safe approach to promoting ICD and activating anti-tumor immunity for combinatorial immune checkpoint inhibition therapies against vision and life-threatening ocular melanomas.

This study was supported by research funding from the Intramural Grants Program, Auburn University Research Initiative in Cancer (AURIC), and start-up funds from the Department of Pathobiology, College of Veterinary Medicine, Auburn University.