bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucocorticoid signaling and regulatory T cell collaborate to maintain the hair follicle stem cell niche
Authors: Zheng Ye , Liu Zhi , Hu Xianting , Liang Yuqiong , Yu Jingting , Shokhirev Maxim N
Published: January 2023

The maintenance of tissue homeostasis in steady state or under stress is dependent on the proper communication between the stem cells and the supporting cells in their microenvironment or “niche”. In addition to promoting immune tolerance, regulatory T cells (Tregs) have recently emerged as a critical component of the stem cell niche in the hair follicle (HF), injured muscle, bone marrow, and small intestine to support stem cell differentiation or maintain their quiescence. How Treg cells sense the dynamic signals in the niche environment and communicate with stem cells during tissue regeneration is largely unknown. Here, by using HF as a model, we uncover a hitherto unrecognized function of steroid hormone glucocorticoid that instructs skin-resident Treg cells through glucocorticoid receptor (GR) to facilitate hair follicle stem cell (HFSC) activation and HF regeneration. Ablation of GR signaling in Tregs blocked depilation-induced hair regeneration and natural hair growth without affecting Treg’s immune suppressive function. Mechanistic study revealed that GR signaling induces skin-resident Tregs to produce TGF-b3, which directly activates Smad2/3 in HFSCs and facilitates HFSC activation and proliferation. Our study identifies a novel crosstalk between skin-resident Tregs and HFSCs mediated by the GR/TGF-b3 axis, highlighting a new avenue to manipulate Tregs to support tissue regeneration.

Supported by NIH R01-AI107027 NIH R01-AI1511123 NIH R21-AI154919 NIH S10-OD023689
Novel SLC5A6 Mutations Lead to B Lymphocyte Maturation Defects with Metabolic Abnormality Rescuable by Biotin Replenishment
Authors: Hsieh Chu-Han , Chen Peng-Chieh , Shieh Chi-Chang
Published: January 2023

Primary immunodeficiencies (PIDs) comprise inherited genetic mutations with a wide spectrum of clinical immune manifestations. We enrolled a seven-years-old female patient whose clinical laboratory tests showed very low levels of all five isotypes of immunoglobulins but with normal numbers of T cells and B cells. By using whole exome sequencing (WES) on the patient and family members, we identified a novel compound heterozygous gene mutations (termed SLC5A6 mutations) in the patient and her deceased elder sister. We successfully established a CRISPR-Cas9 mouse model bearing the same mutation gene from our patient and provide a further chance to investigate detailed mechanism underlie primary immunodeficiency. We found that biotin deficiency because of SLC5A6 mutations lead to defective B cell maturation and antibody insufficiency. We also found that aberrant cellular metabolism profile due to biotin deficiency restrain the efficiency of plasma cell formation and functions. Furthermore, biotin replenishment can not only rescue cellular TCA cycle but also ameliorate immunodeficient phenotypes including antibody insufficiency in patient and in our mouse model. Our results demonstrated the pivotal role of metabolic reprogramming in B cell terminal differentiation and indicated that SLC5A6 is a causative gene for immunodeficiency.
HIV gp120-specific memory develops from pre-existing memory and naive B cells following vaccination in humans
Authors: Kobie James , Piepenbrink Michael , Fucile Christopher , Bunce Catherine , Man Li-Xing , Liesveld Jane , Rosenberg Alexander F , Keefer Michael , Basu Madhubanti
Published: January 2023

The most potent and broad HIV envelope (Env)-specific antibodies often when reverted to their inferred germline versions representing the naïve B cell receptor, fail to bind Env suggesting that the initial responding B cell population is not exclusively comprised of a naïve population, but also a pre-existing cross-reactive antigen-experienced B cell pool that expands following Env exposure. Previously we isolated gp120-reactive monoclonal antibodies (mAbs) from participants from HVTN105, a phase I trial testing AIDSVAX B/E (gp120 protein) combined with a DNA immunogen. Using MiSeq-based deep sequencing VH-lineage tracking we identified several of these mAb lineages in pre-immune peripheral blood. Additionally, we sought to characterize the gp120-specific B cell repertoire prior to immunization, and the features of those pre-immune B cell clonal lineages that ultimately respond to the vaccine. Several of these pre-immune lineages also persisted in the bone marrow, including CD138+ long-lived plasma cell compartment, ~7 months after the final vaccination. Greater gp120 binding activity was observed from the non-naïve pre-immune VH members than their germline VH versions. The majority of the pre-immune gp120-reactive lineages included IgM, however IgG and IgA members were also predominant prior to vaccination. Additionally, the majority of the pre-immune lineage members exhibited somatic hypermutation prior to vaccination, suggesting that the vaccine-induced gp120-specific antibody lineages originated from both naïve B cells and cross-reactive memory B cells.

Supported by grants from the NIH (R01AI117787, R21AI116285).
Sterile liver inflammation and T cell-intrinsic T-bet expression drive the formation of Tissue Resident Memory CD4 T cells that protect against systemic Salmonella infection
Authors: Depew Claire , McSorley Stephen J
Published: January 2023

Infection with Salmonella enterica serovar Typhimurium (ST) can cause invasive Non-Typhoidal Salmonellosis (iNTS), a systemic disease which kills about 150,000 people annually. In a mouse model of systemic salmonellosis, hepatic tissue resident memory (TRM) CD4 T cells provided protection against subsequent infection. However, the signals required to generate these protective CD4 T cells remain poorly defined. To interrogate the process of hepatic CD4 TRM formation we developed a simple Salmonella-specific transfer system. ST-specific (SM1) TCR transgenic CD4 T cells were activated in vitro and adoptively transferred into C57BL/6 mice undergoing acetaminophen overdose. Sterile liver inflammation enhanced the formation of hepatic TRM SM1 T cells which expressed increased levels of T-bet compared to circulating memory cells. Subsequent mixed bone marrow chimera experiments substantiated the requirement for T-bet in hepatic TRM formation as T-bet-deficient CD4 T cells were deficient in resident memory. Bulk RNAseq analysis of hepatic CD4 TRMs uncovered a variety of cytokine and chemokine receptors that correlated with hepatic tissue residency. Using a combination of gene-deficiency and in vivo neutralization approaches, efforts are ongoing to uncover key components of sterile hepatic inflammation that drive CD4 TRM development. Understanding this process is critical to understanding the process of CD4 TRM development and will be foundational for the development of an effective vaccine against iNTS.

Supported by NIAID R01AI139410, NIH T32 Animal Models of Infectious Diseases
Meningeal lymphatic drainage promotes T cell responses against Toxoplasma gondii but is dispensable for parasite control in the brain
Authors: Kovacs Michael , Cowan Maureen N , Still Katherine M , Batista Samantha J , Babcock Isaac , Sibley Lydia , Sethi Ish , Harris Tajie H
Published: January 2023

Toxoplasma gondii is an intracellular protozoan parasite that causes chronic brain infection in a wide range of mammalian hosts. Animal studies have demonstrated that continuous T cell recruitment to the brain is necessary for parasite control. However, it has remained unclear how T cells outside the central nervous system sense and respond to brain-derived microbial antigen. Here, we test the hypothesis that the newly described meningeal lymphatic system promotes T cell immunity during T. gondii brain infection. We find that chronic brain infection is associated with significant expansion of parasite-specific T cells in the cerebrospinal fluid (CSF)-draining deep cervical lymph nodes. T cell activation at this site is correlated with parasite burden in the brain and peaks during the late stages of chronic infection. Flow cytometric analysis of CSF reveals a population of activated dendritic cells that is not present in the CSF of naïve mice. Mature dendritic cells are also mobilized in the meninges, specifically in areas where CSF protein can be sampled. Disrupting meningeal lymphatic drainage by ligating the collecting vessels leads to impaired T cell responses in the deep cervical lymph nodes. Surprisingly, in spite of reduced T cell activation and IFN-γ production at this site, T cell responses in the brain remain intact, likely due to ongoing antigenic stimulation in lymph nodes that drain non-central nervous system tissue. Overall, we provide evidence that meningeal lymphatic drainage supports robust parasite-specific T cell responses in the deep cervical lymph nodes. Nonetheless, we find that drainage of central nervous system material to this site is dispensable for host-protective T cell responses in the brain.

Supported by grants from NIH (5T32AI007496, 5T32GM007267, R01NS091067, R01NS112516, R56NS106028, F30AI154740)
Selective Reprogramming of Peritoneal Macrophages by IFN-γ During Acute Toxoplasma gondii Infection
Authors: Martin Andrew Thomas , Yarovinsky Felix
Published: January 2023

Host resistance to intracellular pathogens is dependent on the pleiotropic cytokine interferon gamma (IFN-γ) which activates myeloid cells, such as macrophages, and results in pathogen clearance. Macrophages safeguard most regions of the body participating in tissue homeostasis, disease surveillance, pathogen elimination, and resolution of inflammation. The peritoneal cavity is patrolled by two unique subsets of resident macrophages: monocyte-derived small peritoneal macrophages (SPMs) and embryonically-seeded large peritoneal macrophages (LPMs). To determine the roles of SPMs and LPMs during intracellular infections, we infected mice intraperitoneally with the intracellular parasite Toxoplasma gondii, a robust driver of IFN-γ-mediated type 1 immune responses. We observed that T. gondii triggers a distinctive response in LPMs and SPMs responding to parasitic infection. Through the use of neutralizing antibodies, recombinant IFN-γ, and mice conditionally lacking expression of the IFN-γ receptor in macrophages, we have shown that the direct effects of IFN-γ are both required and sufficient for selective reprogramming of peritoneal macrophage subsets in response to T. gondii infection. Molecular mechanisms underlying the macrophage subset-specific effects of IFN-γ were further dissected in vivo and in vitro through imaging and omics approaches. Our results revealed the peritoneal macrophage subset-specific responses to IFN-γ that are essential for host resistance to T. gondii.

Supported by the URMC T32 Immunology Training Grant AI00728, NIH R01 GR501266, and the Burroughs Wellcome Fund.
Predicted Cellularity using RNASeq-Based Cellular Deconvolution Differentiates Periprosthetic Joint Infection from Non-Infectious Arthroplasty Failure
Authors: Fisher Cody , Krull Jordan , Bhagwate Aditya , Greenwood-Quaintance Kerryl , Abdel Matthew P , Patel Robin
Published: January 2023

Differentiation of aseptic and septic causes of arthroplasty failure is essential due to their different treatment schemes. We hypothesized that predicted cellularity profiling of sonicate fluid using the RNASeq-based the cellular deconvolution tools CIBERSORTx and ABIS-seq can differentiate between PJI and non-infected arthroplasty failure (NIAF). Profiles were created for 93 sonicate fluid samples (40 from NIAF and 53 from PJI patients) that had been subjected to RNASeq analysis. CIBERSORTx provided 22 total predicted cell types; 12 predicted cellular abundances were differentially expressed in PJI versus NIAF, including increased neutrophils, activated mast cells, and eosinophils (p≤0.0004) and decreased M0 macrophages, M2 macrophages, and Treg cells (p≤0.0001). ABIS-seq provided 17 total predicted cell types; seven predicted cellular abundances were differentially expressed in PJI versus NIAF, including increased neutrophils, Vd2 γδ T cells, and basophils (p-values of <0.0001, 0.0003, and 0.0263, respectively) and decreased nonclassical/intermediate (NC+I) monocytes, mucosal-associated invariant T (MAIT) cells, and myeloid dendritic cells (mDC) (p-values ≤0.0001). Receiver operative characteristic area under the curve (AUC) analysis identified cells types most predictive of PJI (CIBERSORTx: neutrophils [AUC = 94], activated mast cells [AUC = 93]; ABIS-seq: neutrophils [AUC = 88]) and NIAF (ABIS-seq: NC+I monocytes [AUC = 85], MAIT cells [AUC = 80], mDCs [AUC = 81]). PJI and NIAF samples were differentially clustered by principal component analysis using both tools. Overall, cellularity profiling using RNASeq-based cellular deconvolution can differentiate between PJI and NIAF sonicate fluid samples.

Work supported by NIAMS (NIH R01 AR056647). CF was supported by the Mayo Clinic Graduate School of Biomedical Sciences and the Ph.D. Training Grant in Basic Immunology (NIH R25 GM055252 24).
Type I Interferon remodels intrahepatocytic signaling to promote T cell dysfunction during liver stage Plasmodium infection
Authors: Minkah Nana K , Reynolds Laura , Okolo Vera , Kappe Stefan
Published: January 2023

Malaria is caused by Plasmodium parasites and accounted for 627,000 deaths in 2021. Upon deposition into the dermis, Plasmodium parasites traffic to, infect, and develop within hepatocytes before egressing to cause symptomatic erythrocytic infection. We recently reported that liver stage Plasmodium replication induces type I Interferon (IFN-I) signaling that compromises anti-malaria T cells. We now show that this IFN-I-mediated, inferior T cell response is mostly limited to hepatic CD8 T cells and is characterized by diminished cytokine production, impaired homeostatic CD8 T cell survival, and the expression of receptors and transcription factors associated with T cell exhaustion. We set out to identify the cells and molecular events involved in the induction of T cell exhaustion during the transient and non-persistent liver stage Plasmodium infection and observed that infected mice lacking IFNAR expression solely on hepatocytes do not generate significant frequencies of exhausted T cells. Furthermore, transcriptomic analyses of hepatocytes isolated during infection indicate that IFN-I signaling coincides with a remodeling of the hepatocyte transcriptome such that antigen processing and presentation, chemokine transcripts and immunoregulatory receptors and soluble factors are upregulated. We hypothesize that IFN-I signaling reshapes the hepatocyte transcriptome to promote an immunosuppressive liver microenvironment that impairs anti-malaria T cells. Together, our studies represent the first description of a negative role for liver stage IFN-I signaling on anti-malaria adaptive immunity and may necessitate a paradigm shift in the rational design of efficacious anti-malaria whole parasite vaccines.

Supported by 1U01AI42001
Combined analysis of T cell activation and T cell-mediated cytotoxicity by imaging cytometry
Authors: Chanda Monica Kasturi , Shudde Claire E , Piper Taylor L , Zheng Yating , Courtney Adam H
Published: January 2023

Immunotherapies have transformed the treatment of cancer through the development of drugs that allow T cells to recognize and kill malignant cells. With this has come a need to evaluate how these drugs affect T cell function. We developed an imaging cytometry-based method that can simultaneously assess T cell activation and the functional ability of these activated T cells to kill cancer cells. We used the Nur77-GFP reporter, which expresses GFP upon T cell receptor signaling, as a readout of T cell activation. We combined this reporter system with an MC38 colon adenocarcinoma cell line expressing a TCR-specific antigen that is used as a model tumor antigen (TA). We show that in T cell-cancer co-culture systems, we are able to quantify the number of activated cytotoxic CD8+ T cells and their ability to recognize and kill cancer cells by imaging cytometry. We tested the sensitivity of this platform by quantifying the proportion of antigen-specific activated T cells in co-cultures with varying ratios of unresponsive T cells, mimicking heterogenous lymphocyte populations. To test our system in a pharmacological context, we evaluated the effects of a SRC family kinase inhibitor on T cell activation and cancer cell death. In each case, we were able to quantify the proportion of activated T cells and the extent of TA-specific cancer cell death. Our findings show that the Nur77-GFP reporter system combined with imaging cytometry is a fast and reproducible system to evaluate how different treatment conditions influence TA recognition by T cells using T cell-cancer co-cultures. The simultaneous analysis of T cell activation with T cell-mediated cancer cell death can be used to rapidly assess immuno-oncology drug candidates and T cell-based therapeutics.

Supported by The Elsa U. Pardee Foundation (AHC), the V Foundation Scholar Award (AHC), the Cancer Biology Training Program T32CA009676 (MKC), the Pharmacological Sciences Training Program T32GM140223 (CES), and a Charles W. Edmunds Pharmacology Fellowship (YZ). This study was also supported by resources provided by the U-M Flow Cytometry Core and the U-M Rogel Cancer Center (P30CA046592).
Use of novel fluorescent dyes enables in depth analysis of checkpoint inhibitors CTLA-4 (CD152) and PD-1 through spectral flow cytometry
Authors: Ellsworth Jessica , DeFelippi Kendall , Strenger-Smith Jazmine , Zahner Sonja
Published: January 2023

PD-1 and CTLA-4 (CD152) are prominent checkpoint inhibitors in T cell responses and their inhibition has emerged as a successful route to boost anti-tumor immune responses in cancer patients. Clinical trials involving CAR-T cell therapy with anti-CTLA-4 and PD-1 antibodies seem to be improving the tumor directed T cell response.

Regulation of surface vs cytoplasmic CTLA-4 pools is crucial to balancing stimulatory and inhibitory signals in T-cell responses. Surface CTLA-4 contributes to tumor escape by inhibiting T cell responses, and upregulation of CTLA-4 expression from the cytoplasm to the surface is highly regulated. Therefore, it might be of interest to monitor these CTLA-4 pools as this might indicate the efficacy of CAR-T cell therapeutic approaches. Here we show how novel fluorescent dyes enable the composition of a spectral flow cytometry panel to interrogate the presence of surface versus cytoplasmic CTLA-4 pools and the dynamics of PD-1 expression in a single panel. Combining these parameters into a single panel allows for less workflow steps and permit conservative sample use.
Advancements in single cell multiomic profiling of antigen-specific T cells with dCODE Dextramer&[trade] (RiO) and BD® AbSeq Reagents on the BD Rhapsody&[trade] Single-Cell Analysis System
Authors: Sakofsky Cynthia , Jacobsen Kivin , Lopez-Salmeron Vadir , Nakamoto Margaret , Brix Liselotte , Ayer Aruna
Published: January 2023

The advancement of immunotherapies and drug development relies on the characterization of antigen-specific T cells. However, due to their low frequency among T cells and their inherent weak affinity to bind known MHC-peptide complexes, detection of these cells has been difficult. Furthermore, pairing this information with the corresponding variable (V), diversity (D), and joining (J) [V(D)J] sequences of the antigen-specific T cell receptors has also been challenging due to hurdles in sequencing these large regions, particularly at the single-cell level. Here, we have expanded our previous work of combining two powerful technologies, Immudex dCODE Dextramer™ (RiO) Reagents and the BD Rhapsody™ Single-Cell Analysis System, to detect and characterize low-frequency antigen-specific T cells, including the full sequences of the V(D)J gene segments of the T cell receptors, as well as profile transcriptome and protein expression. Specifically, thousands of sorted PBMCs were multiplexed to provide high-throughput detection of individual antigen-specific CD8+ T cells in combination with the corresponding full V(D)J sequences of the T cell receptors. In addition, we simultaneously obtained gene expression data for over 400 immune-related mRNAs as well as cell phenotypes using a panel of 30 cell surface BD® AbSeq Protein Markers. Together these data can be used to define T cell phenotypes associated with T cell activation states, alongside antigen specificity of enriched CD8+ dextramer+ cells from PBMC populations. This study showcases the importance of multiomic analysis for high-resolution T cell profiling that has broader implications and utility in immuno-oncology, infectious diseases and autoimmunity.
A Rapid, Calcium-independent Mojosort &[trade] Dead Cell Removal Kit for Improved Live Cell Enrichment and Downstream Application
Authors: Zhang Hong , Ruiz Jen , Yamamoto Kenta , Taylor Kevin , Wiethe Carsten , Yang Xifeng
Published: January 2023

High sample cell viability is crucial for obtaining high-quality data for various downstream applications. Commonly used methods include density centrifugation, flow cytometry sorting, and Annexin V-based magnetic cell separation. However, above entioned methods have several limitations that ultimately leads to fewer live cell recovery. Here we present MojoSort™ dead cell removal kit, which relies on Ca2+-independent, Phoshatidylserine (PS)-binding for dead and apoptotic cell removal. Unlike Annexin V that requires specialized, high Ca2+-containing buffers that can potentially induce cytotoxicity, MojoSort™ dead cell removal kit can be performed in the buffer with or without Ca2+ that also contain chelators such as EDTA. This enables 20% improved live cell recovery compared to other commercially available, Annexin V-based magnetic cell separation kits, while demonstrating 90–95% live peripheral blood mononuclear cell (PBMC) purity post-enrichment from 50% starting viability. Single-cell cellular Indexing of transcriptomes and epitopes by sequencing (CITE-seq) demonstrated effective removal of a distinct dead cell cluster comprised of cells with high mitochondrial gene read counts, while preserving all common PBMC lineages and surface marker expression levels. In summary, MojoSort™ dead cell removal provides a rapid, effective live cell enrichment option that overcomes shortcomings associated with other routinely used methods for downstream applications that require high sample viability for success.
“Therapeutic targeting of tumor necrosis factor like weak inducer of apoptosis (TWEAK) in psoriasis”
Authors: Gupta Rinkesh Kumar , Gracias Donald , Figueroa Daniela Salgado , Miki Haruka , Miller Jacqueline , Fung Kai , Ay Ferhat , Burkly Linda C , Croft Michael
Published: January 2023

Excessive activity of TNF and IL-17 in keratinocytes can drive psoriatic skin inflammation and their targeting is highly advantageous in psoriasis patients, but whether these cytokines act with other inflammatory molecules is unknown. Previously, we demonstrated with knockout mice that the TNF-related cytokine TWEAK (TNFSF12) was a major driver of skin inflammation in the imiquimod model of psoriasis. Here, we found that mice carrying a deletion of TWEAK’s receptor, Fn14 (TNFRSF12A), specifically in keratinocytes, exhibited reduced psoriasis features, including less epidermal hyperplasia and poor expression of genes associated with human psoriasis. Further, RNA-seq analysis in human keratinocytes demonstrated that TWEAK strongly synergizes with either TNF or IL-17A in upregulating the transcripts for psoriasis-associated chemokines and cytokines, implying that TWEAK co-operates with TNF and IL-17 to enhance feedback inflammatory activity. Correspondingly, administration of anti-TWEAK antibody demonstrated therapeutic efficacy in the psoriasis mouse model and was equally as effective as antibodies to IL-17A or TNF in reducing clinical and immunological features of psoriasis-like skin inflammation, while co-neutralization of TWEAK with either cytokine had no additional therapeutic effect, reinforcing the conclusion that all three cytokines act together. We conclude that TWEAK blockade could be another potential therapeutic avenue in patients with psoriasis that might be equally as useful as blocking TNF and IL-17. Future studies targeting TWEAK in clinical trials for psoriasis may then be warranted.

Supported by NIH grant AR072640
DRAK2 is a promising target to inhibit GvHD without negatively impacting graft reconstitution after bone marrow transplant
Authors: Duggar Marygrace S , Wehenkel Marie , Vogel Peter , McGargill Maureen Ann
Published: January 2023

Graft versus host disease (GvHD) is the most common adverse event after allogeneic bone marrow transplant (BMT). GvHD and the immunosuppression required to treat it limit the desired graft versus leukemic effect and are associated with significant morbidity and mortality. A treatment that selectively controls the harmful immune response during GvHD, without compromising the response to pathogens or tumors, would be transformative. Previously, we demonstrated that mice deficient in Drak2 (Drak2−/−) are resistant to disease in multiple models of T cell-mediated autoimmunity but maintain effective responses to pathogens and tumors. Since similar immune pathologies mediate GvHD and autoimmunity, we sought to investigate whether loss of Drak2 reduced GvHD, while maintaining effective immunity to pathogens. Thus, we utilized a murine model of acute GvHD (aGvHD) in which lethally irradiated mice received MHC-mismatched bone marrow and purified T cells that were either Drak2+/+ or Drak2−/−. We found that mice given MHC-mismatched bone marrow along with Drak2−/− T cells had less severe aGvHD compared to mice receiving Drak2+/+ T cells, as demonstrated by lower clinical scores. Interestingly, mice given Drak2−/− T cells along with MHC-mismatched bone marrow had increased proportions of regulatory T cells and decreased effector T cells compared to mice given wildtype T cells. These data suggest that Drak2 expression within T cells contributes to GvHD development, and therefore is a promising target in the treatment of GvHD. Ongoing studies are investigating whether the absence of Drak2 influences the effective repopulation of a functional immune system following BMT.

Funding provided by ALSAC and the St. Jude Graduate School of Biomedial Sciences
Seeking the Sertoli cell complement inhibitory signature to prolong transplant survival
Authors: Washburn Rachel , Kaur Gurvinder , Chilton Beverly , Reilly Brian D , Dufour Jannette
Published: January 2023

Clinical transplantation can treat various illnesses, although without the use of toxic immunosuppressants transplanted tissue is rejected by the immune system. Complement, an enzymatic cascade that leads to cytolysis by insertion of the membrane attack complex (MAC), is a significant component in transplant rejection. Interestingly, Sertoli cells (SC) survive long-term after transplantation across immune barriers without immunosuppression. This study used allotransplant (between same species) and xenotransplant (between different species) models to investigate the role of complement inhibition in SC extended survival. Since SC were resistant to in vitro complement killing, mouse or pig SC were transplanted into mice or rats, respectively. Grafts were collected and analyzed for MAC. MAC was not detected on SC transplants, indicating that SC inhibit complement before MAC deposition, possibly through expression of complement inhibitory proteins (CIP). Bioinformatic analyses of mouse or pig SC identified expression of 13 CIP. After expression of two key CIP, CD46 and CD55, were knocked down, SC were killed by complement in vitro. Taken together, these data indicate that the SC complement inhibitory signature is critical for SC graft survival. Understanding SC complement inhibition is important as it could lead to improved transplant survival with a decreased requirement of harsh immunosuppressants, making this procedure more attainable for patients.

Supported by grants from the CH foundation, the South Plains Foundation, and the Mary Lou Clements-Mann Scholarship
Sex-specific T cell behavior drives differential immune responses in mouse glioblastoma models
Authors: Lee Juyeun , Lathia Justin D
Published: January 2023

Glioblastoma (GBM) is the most common primary malignant brain tumor and has a median survival of 12–18 months with current standard-of-care of therapy. GBM sex differences in incidence and overall survival, with males experiencing a higher incidence and a worse prognosis compared to females. However, the role of immune cells in GBM sex differences outside of myeloid cells remains poorly understood. Using a syngeneic mouse GBM model, we recapitulated the sex differences observed in patients, with shortened survival in male hosts compared to female hosts. These findings were not recapitulated in immuno-deficient mouse strains such as NSG and RAG1KO mice, suggesting a role for immune cells, specifically T cells, in GBM sex differences. Flow cytometry analysis of tumor-infiltrating leukocytes revealed more T cells in female tumors, whereas male tumors were enriched in macrophages. Additionally, more T cells in male tumors exhibited high levels of inhibitory receptors, whereas most female T cells were more functional as measured by expression of anti-tumor cytokines such as TNF, IFN-gamma, and granzyme B. A bone marrow chimera model revealed that male T cells retained their phenotype in female hosts, whereas female T cell behavior was affected by the male environment. Furthermore, female T cells are more proliferative, with elevated expression of integrins compared to male T cells. Lastly, we found that males have more progenitor exhausted T cells, which led to better response to anti-PD1 treatment. Collectively, these results suggest that both cell-intrinsic and cell-extrinsic factors regulate T cell activity in a sex-specific manner, providing insights to develop sex-specific therapeutic approaches.

Supported by grant from NIH (P01 CA245705)
Senescent cell-derived extracellular vesicles are critical elements in senescence surveillance by recruiting antigen-presenting cells
Authors: Ziglari Tahereh , Claudio Natalie , Guo Zihan , Pucci Ferdinando
Published: January 2023

Cellular senescence is a tumor suppressor mechanism that arrests cell cycle and initiates immune-mediated removal of senescent cells, a process known as senescence surveillance (SS). Senescent cells interact with their microenvironment through senescence-associated secretory phenotype (SASP). Current knowledge about the SASP has focused on soluble elements like cytokines. Extracellular vesicles (EVs) are membrane-bound, nanometer-sized particles that are emerging as a novel class of SASP factors. Whether EVs play a role in SS is unknown. Elucidating the role of EVs in SS will open new avenues for therapeutically targeting cellular senescence. In order to study if and how EVs contribute to SS, we inhibited EV release from senescent cells using dominant-negative mutant Rab35. This approach preserved secretion of soluble SASP elements while decreasing >95% the release of EVs from senescent cells. C57BL6 mice were challenged with EV-inhibited senescent cells and their numbers at the site of injection as well as immune infiltrates were quantified. Interfering with EV release led to the reduction of antigen-presenting cells including dendritic cells, macrophages, and B cells, which may contribute to the observed persistence of senescent cells. Interestingly, EV-inhibition reverted cellular senescence, promoted proliferation and tumor formation. Proteomic analysis revealed 35 proteins that were exclusively present on senescent cell-derived EVs, compared to those from their non-senescence counterparts. These results suggest that EVs have critical role in SS possibly via signaling to immune cells. Future work will explore whether T cells respond to EV-bound senescent cell antigens.

Integrated Training in Quantitative and Experimental Cancer Systems Biology, 5T32CA254888-02 and CRUK/OHSU A29681
The nucleic acid sensor RIG-I as an inducer of anti-tumor response
Authors: Fraile-Bethencourt Eugenia , Khou Sokchea , Anand Sudarshan
Published: January 2023

The immune microenvironment plays a critical role in tumor progression. Recently, approaches to target Pattern Recognition Receptors (PRRs) have emerged as a promising strategy to enhance immune recognition of tumors. Among the PRRs, those which recognize DNA and RNA in the cells are unique in their ability to respond to both pathogenic and self-nucleic acids that are generated as a result of aberrant cell division/transcription. We previously showed that targeting the DNA sensor three prime exonuclease 1 (TREX1) enhanced immune responses in mouse tumor models. Here we show that the RNA sensor Retinoic acid Induced Gene 1 (RIG-I) acts as a robust immune activator across cancer types. According cBioportal data, expression of RIG-I seems to be a prognostic factor for progression free survival. To functional evaluate RIG-I response in tumor models, we used a hairpin RNA derived from H1N1 sequence. Interestingly, activation of RIG-I increased expression of TREX1 revealing a potential crosstalk between the two pathways. Both breast and colorectal tumor cell lines produce IFN-I after RIG-I activation. Consistently, RNAseq showed upregulation of IFN-I response genes and proinflammatory cytokines across different cell lines. Activation of RIG-I in the tumor cells decreased tumor burden with a concomitant broad cellular and molecular anti-tumor response. Thus, results showed an increase in NK and DC population, while the transcription profile was upregulation of innate immune response genes. Interestingly, tumor growth and NK results were consistent when tumor was implanted in immunocompromised mouse model. Taken together, our findings illustrate how tumor cell RIG-I is a potent inducer of cellular and molecular immune responses.
Diminished noncanonical NF-κB signaling induces colitis-associated colorectal cancer susceptibility upon de-differentiation of epithelial cells
Authors: Morrison Holly , Rowe Audrey , Eden Kristin , Baumgarner Katherine , Brown Stephan L , Holl Eda K , Allen Irving Coy
Published: January 2023

Patients with a medical history of Inflammatory Bowel Disease are predisposed to developing colitis-associated colorectal cancer (CAC), with greater than 20% developing CAC within 30 years of disease onset. CAC is a form of inflammation-induced tumorigenesis, where unregulated inflammation causes cells in the colon to accumulate mutations poising them for neoplasia. We demonstrate that NF-κB inducing kinase (NIK) is a critical regulator of intestinal epithelial cell (IEC) regeneration. We hypothesize that IECs exposed to chronic inflammatory conditions mitigated by dysregulated noncanonical NF-κB signaling are most prone to mutagenesis and cancer-initiating events termed Top-down tumorigenesis. In CAC, NIK depletion results in the accumulation of mature IECs that are increasingly susceptible to mutation and neoplastic transformation. This suggests that terminally differentiated IECs dedifferentiate into a stem-like phenotype. Our work has clinical relevancy, where human CAC biopsy samples have diminished noncanonical NF-κB signaling and NIK (MAP3K14) gene expression. Murine models with NIK deletion in IECs have increased susceptibility to inflammation-induced tumorigenesis following the chemical induction of AOM/DSS to induce colitis. Our mechanistic studies evaluating crypts demonstrate accumulation of mature, non-dividing IECs, which are more susceptible to mutations and malignant transformation. Here, we present a novel role for noncanonical NF-κB signaling in regulating intestinal epithelial cell turnover in protecting against CAC development. Focusing on the key niche signaling interplay following noncanonical NF-κB dysregulation gives greater insight into the CAC tumor microenvironment.

Supported by grants from the iTHRIVE Pilot Translational and Clinical Studies Program, VCOM Center for One Health Research, Virginia Tech Carilion Research Acceleration Program, and the Virginia-Maryland College of Veterinary Medicine.
Egfl6 promotes ovarian cancer progression by inducing the immunosuppressive functions of tumor-infiltrating myeloid cells.
Authors: Cascio Sandra , Sinno Sarah , Bai Shoumei , Coronnello Claudia , Vlad Anda , Buckanovich Ronald J
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are critical negative regulators of immunity in cancer. Understanding factors which regulate these cells could result in the identification of new approaches to enhance anti-tumor immunotherapy. One such factor is epidermal growth factor-like 6 (EGFL6), a secreted factor known to promote cancer stem like cell migration and regulate cancer cell differentiation. We found that mice which overexpress Egfl6 have an increased numbers of granulocytes and monocytes in both the bone marrow and spleen. In vitro and ex-vivo analysis indicated that EGFL6, via binding with beta integrins and activation of SYK and ERK signaling, (i) acts as a chemotactic factor for myeloid cells migration and (ii) promotes their differentiation toward a suppressive state. Suggesting an important role in promoting an immunosuppressive tumor microenvironment (TME), using two syngeneic mouse models of ovarian cancer, we found that expression of Egfl6 in tumor cells resulted in increased accumulation of intra-tumoral MDSCs and TAMs and fewer cytotoxic CD8+ T cells. This was associated with increased tumor growth and shortened animal survival. Gene expression profiling of tumor infiltrating myeloid cells indicated that Egfl6 induced the expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Moreover, in an immune ‘hot’ tumor model, EGFL6 completely inhibited response to a-PD-L-1 therapy. Combined our data show that EGFL6 induces the recruitment of myeloid cells into the ovarian TME and subsequently promotes their immunosuppressive functions. This suggest EGFL6 is a potential novel therapeutic target to enhance response to immune therapy in OvCa patients.

Supported by Ovarian Cancer Research Alliance
Glycoprotein-NMB (GPNMB) is Pro-Tumorigenic in Lymphangioleiomyomatosis (LAM)
Authors: Gibbons Erin , Taya Manisha , Hammes Stephen
Published: January 2023

Lymphangioleiomyomatosis (LAM) is an estrogen-sensitive lung disease found almost exclusively in women. LAM is characterized by the hyperproliferation of smooth muscle cells creating small tumors throughout the lungs, resulting in the formation of large cysts that replace normal alveolar space. Growth of these tumors and progression of the cyst development leads to pulmonary function loss, and sometimes subsequent lung transplantation. However, LAM recurs after transplantation and LAM cells can be found circulating in body fluids suggesting a metastatic nature of the disease. Due to LAM’s estrogen sensitivity, female specificity, and metastatic nature, we previously proposed that LAM cells originate from the uterine myometrium. We therefore designed a uterine-specific TSC2-null mouse model where all of the mice generate uterine tumors characteristic of LAM and half develop lung metastases. Using RNASeq analysis of uterine tissue from this mouse model, when focusing on genes regulated by estrogen and TSC2, we discovered increased expression of melanocytic markers, including Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB), which is known to be oncogenic in other cancers. GPNMB was not only highly expressed in our mouse model, it was also expressed in TSC2-null cell lines, and LAM patient lungs and uteri. Interestingly, serum GPNMB is a potential biomarker, as LAM patients have higher serum GPNMB levels compared to healthy controls. Knocking down GPNMB expression by siRNA decreased proliferation, migration, and invasion in TSC2-null cells. Further, knocking out GPNMB in TSC2-null cells using CRISPR/Cas9 decreased xenograft tumor growth in mice and GPNMB is a potential therapeutic target for LAM.

Supported by grants from NIH (R01 CA193583, T32HL066988), the LAM Foundation, and DOD
Defining myeloid plasticity and heterogeneity in immunotherapy response
Authors: Dinh Huy , Golfinos Athena , Wang Wei , Mergaert Aisha , Lambert Paul
Published: January 2023

Like many cancer types, Head and neck cancers (HNC) have a <20% response rate to immune checkpoint inhibitor (ICI) therapy, making it imperative to learn what causes high treatment resistance. In addition, dissecting the tumor immune heterogeneity and plasticity is crucial to understand and overcome these challenges. Recent scRNA-seq studies have extensively characterized lymphocyte cells in HNC. However, myeloid cells are not well characterized, despite composing many in tumors and exhibiting pro- and anti-tumoral phenotypes. Here we show a comprehensive cross-species characterization of myeloid cells in HNC humans and mouse models and how they influence ICI response. To this end, we generated scRNA-Seq of immune cells in tumors from two HNC mouse models with different responses to ICI. Our comparative analysis identified critical changes in the myeloid compartment in responder and non-responder mice receiving ICI (combined anti-PD1 + anti-CTLA4) therapies. Examples are macrophage and dendritic cell subsets expressing CXCL9 and PDL1, two predictors of ICI response reported in HNC clinical trials and recently published work. Our bioinformatics approaches also defined ligand-receptor cell-cell interactions between myeloid and T cells that influence downstream gene expression networks as potential underlying mechanisms and actionable targets for ICI response. Interestingly, we observed a high concordance between mouse and human myeloid cells from ours and publicly available scRNA-Seq data. Our data and analysis provide the first translational mouse-to-human atlas of myeloid cells in HNC, providing a way to inform biomarkers and immunotherapeutic targets for potential ICI therapy success in HNC and other cancer types.

This research was supported by the University of Wisconsin Head and Neck SPORE grant CEP award (P50DE026787 to H.Q.D)
Cellular avidity as a novel biomarker for candidate selection of cell-based immunotherapies
Authors: Eberlein Jens , Moser Justin , Zhong Zhou , Bailey Keith A , Hoang Song-My , Schultz Aric J , Candelli Andrea , Reijmers Rogier M , Singleterry Will
Published: January 2023

Cellular immunotherapies are increasingly complex in design. Affinity readouts, i.e., the measurements of the strength of single receptor-ligand interactions, are frequently used to assess the binding of chimeric antigen receptors (CARs) or T cell receptors (TCRs) to their ligand. However, affinity is a poor predictor of effector function of the engineered downstream product. Therefore, new tools are needed to evaluate the binding strength of CAR and TCR-transgenic cells to their cognate targets in a more biologically relevant context.

Recent studies demonstrated the utility of measuring cellular avidity as a novel biomarker for identifying and developing potent and safe immunotherapies. Unlike affinity, cellular avidity is driven by the overall strength of dynamic surface interactions between effector cells and their targets by integrating receptor density, the sum of individual affinities, and engagement of the multitude of co-receptors within the immunological synapse.

Here, we show that increased specific avidity, i.e., TCRs with the strongest antigen binding and the lowest background, correlated with improved effector function both in vitro and in vivo. For CAR-T, higher cellular avidity was significantly correlated with improved tumor control in in vivo murine models, but also associated with toxicities in patients, suggesting fine-tuning of cell therapies to the desired avidity for ideal function is needed.
TIM-3+ T regulatory cells alter the lymphoid compartment in cold and Hot solid tumor Model.
Authors: Banerjee Hridesh , Nieves-Rosado Hector M , Murter Benjamin , Kane Lawrence P
Published: January 2023

There is a progressive TIM-3+ T reg increase in solid tumors as the tumor progress. W have found that knocking out the ability of T reg to induce TIM-3 lead to delayed progression of tumors in the TIM-3 conditional knockout mice upon both hot (MC38) and cold (B16F10) syngeneic tumor challenges. We are employing our conditional T reg specific TIM-3 Knockout mouse to study if presence or absence of TIM-3 on T reg cells within these tumor models. Preliminary data has shown that our conditional Tim3 knockout T reg cells are found at a much lower frequency, and have an altered suppressive phenotype within the microenvironment of these tumors. We are currently investigating the other changes in the different subsets of lymphoid compartment in tumors. These data suggest that altering the expression of TIM-3+ on T reg has the potential to act as an effective target to alter tumor microenvironment.

Targeting TIM-3+ T reg may therefore be attractive model to study, along with other combination immunotherapy to increase the success in immunotherapy on patients who develop resistance to primary immunotherapy.
Identification of adjuvants for γδ T cells in response to a novel M. tuberculosis vaccine antigen
Authors: Phelps Kathleen , Xia Mei , Rasi Valerio , Fox Christopher , Petrovsky Nikolai , Carter Darrick , Hoft Daniel
Published: January 2023

γδ T cells are ideal effector cells against infectious diseases and cancer. They reside in blood, lymphoid organs, and mucosa, readily available for host defense. γδ T cells develop memory responses independent of MHC presentation. They perform immune functions such as cytolysis, cytokine production, antigen presentation, dendritic cell maturation, and promotion of B cell and T cell responses. Our lab reported that γδ T cells responsive to M. tuberculosis-specific 6-O-methylglucose-containing lipopolysaccharides (mGLP) inhibit intracellular mycobacterial growth. Preliminary data from NHP studies indicate that vaccination with mGLP and IL-2 greatly reduces Mtb burden post-challenge. IL-2 is not translatable for adjuvant use in humans. Therefore, novel adjuvants for γδ T cells are needed to develop optimal vaccine strategies. We are investigating adjuvant candidates to determine their ability to induce optimal activation and expansion of γδ T cells with mGLP or the phosphoantigen HMBPP. To screen adjuvants, PBMCs are incubated with antigen and adjuvant for 7 days, then analyzed for expansion of γδ T cells. Initially few adjuvants promoted γδ T cell expansion in response to HMBPP and none to mGLP. When matured monocyte-derived DCs were added, multiple adjuvants promoted γδ T cell expansion in response to HMBPP and mGLP stimulation without IL-2. We will determine mechanisms involved in novel adjuvant activity, whether adjuvant and mGLP-expanded γδ T cells inhibit intracellular mycobacterial growth, identify cytokines involved in candidate adjuvant effects, characterize cell surface markers upregulated on DC and γδ T cells, and test the best candidate adjuvant(s) with mGLP in NHP trials.

Supported by Gama Delta R01 AI048391