bluuebunny/crossref_metadata_2025 huggingface dataset:
High-dimension flow cytometry reveals comprehensive deviations in immunophenotypes associated with non-response to melanoma immunotherapies
Authors: Woods David M , Laino Andressa , Winters Aidan , Alexandre Jason , Weber Jeffrey , Chattopadhyay Pratip
Published: January 2023

Immunotherapies have revolutionized the treatment of many cancers, but most patients fail to respond. To investigate immune phenotypes associated with patient response, we assessed 68 unique peripheral blood markers using high-dimension flow cytometry. Samples from 41 metastatic melanoma patients treated with αPD1 or αCTLA4 and 12 healthy donors (HD) were evaluated. To avoid a loss of higher order data, we used a novel computational approach, CytoBrute, to analyze the data through semi-comprehensive Boolean gating. Lineages (e.g. CD3+CD4+) were evaluated for all possible combinations for up to 15 markers. In αPD1 treated patients 1,051 immune signatures differentiated patient response at baseline with a p-value <0.05, while 823 differentiated αCTLA4 treated patients. Only eight signatures overlapped between the two therapies. Signatures included both low (e.g. CD4+CD45RO+CD278−) and high-dimension immunophenotypes (e.g. CD14+CD33+CD11C+CD163+PDL2+41BBL+CD86+CD40+PDL1−CD66B−CD80−GAL9−CD15−CD19−OX40L−). A total of 2,895 signatures significantly differentiated HD versus patients, of which 270 significantly differed (p<0.05) based on patient response. Samples from non-responding patients less closely resembled HD relative to responding patient in 205 of these 270 signatures (p<1e-8). Comparisons of all 102,000 assessed phenotypes showed that non-responding patients had a lesser degree of similarity in immunophenotypes in comparison to responders. Our data demonstrate a novel and powerful approach to interrogating complex immunophenotypes and show that comprehensive immunophenotypic deviations are associated with non-response to melanoma immunotherapies.
The natural human IgG1 mAb Pritumumab targets cell surface expressed vimentin and inhibits tumor growth
Authors: Glassy Mark C , Babic Ivan , Mukthavaram Rajesh , Jiang Pengfei , Nomura Natsuko , Glassy Eric , Kesari Santosh
Published: January 2023

Cancer patients generate tumor-specific B lymphocytes which can be isolated to develop human mAbs against tumor-associated antigens. Pritumumab (also referred to as CLNH-11, CLN-IgG, or ACA-11) is a classic example of a natural human anti-cancer antibody. It is a natural human IgG1 kappa antibody developed by human hybridoma technology, using B lymphocytes isolated from a regional draining lymph node of a patient with cervical carcinoma. Phase II clinical trials with pritumumab have demonstrated an objective response as a therapeutic antibody for glioblastoma. Here we demonstrate pritumumab binds cell surface expressed vimentin, also referred to as ecto-domain vimentin (EDV). Vimentin is an intracellular cytoskeletal protein overexpressed during epithelial-to-mesenchymal transition (EMT), a process integral to cancer cell metastasis. The potential of pritumumab as a therapeutic antibody targeting glioblastoma was evaluated. We demonstrate pritumumab binding to patient glioblastoma cells and antibody binding to these cells induces antibody-dependent cell-mediated cytoxicity (ADCC). Furthermore, pritumumab effectively inhibited glioma tumor growth in a xenograft mouse model. Overall, these data provide pre-clinical validation of pritumumab mAb as a therapeutic for glioblastoma.
Intratumoral delivery of RIG-I agonist induces robust anti-tumor immune responses
Authors: Jiang Xiaodong , Fedorova Olga , Linehan Melissa , Dong Huiping , Pyle Anna Marie , Iwasaki Akiko
Published: January 2023

Recent studies have revealed that cytosolic nucleic acid-sensing pathways such as cGAS-STING and RIG-I-MAVS are involved in cancer biology. How to employ agents or agonists of these pathways to develop new approaches for cancer treatment is a promising direction. In this study, we synthesized a unique, binding-specific RIG-I agonist called Stem Loop RNA (SLR) 14 and evaluated its in vivo anti-tumor efficacy. When tumor volume reached 40–80 mm3, SLR14 was delivered intratumorally, every 2–3 days, for a total of 5–6 doses. Our results show that, after SLR14 treatment, B16 melanoma growth was dramatically blocked and the mice displayed long-term survival. We also observed a significant increase of tumor-infiltrating lymphocytes, and demonstrated that anti-tumor efficacy of SLR14 was T cell-dependent using RAG−/− mice and T cell depletion. Next, we tested anti-tumor efficacy of SLR14 in the mice with immunogenic tumor --YUMMER1.7 (melanoma) or MC38 (colon cancer). Similarly, we also observed a significant delay of tumor growth and extended mouse survival. Moreover, combination treatment with SLR14 and anti-PD1 in these mice led to more remarkable antitumor effects than single treatment. Lastly, we found that SLR14 was mainly taken up by CD45+CD11b+ leukocytes including neutrophils and myeloid cells, suggesting that anti-tumor efficacy of SLR14 is initiated through activation of the RIG-I pathway in innate cells within the tumor microenvironment. Taken together, our data demonstrate that synthetic RIG-I agonist SLR14 is a promising therapeutic agent for a broad spectrum of tumor subtypes. A better understanding of nucleic-acid sensing mechanisms within the tumor microenvironment may yield novel approaches for cancer immunotherapy.
Detection and Quantification of Circulating Tumor Cells in PBMCs or Blood Using Flow Cytometry
Authors: Lei Ming , Ye Peifang , Wang Andy , Li Nancy , Wang Xiaobo
Published: January 2023

Circulating Tumor Cells (CTCs) are cells that have detached from primary tumor tissues or metastases and entered into peripheral blood circulation, led to cancer recurrence and distal metastasis. Evaluation of CTCs conduces to tumor diagnosis, treatment, monitoring, and prognosis. Tumor cell spiking assay was frequently used to evaluate the sensitivity, specificity, accuracy, and repeatability of the methods or systems for enumeration of rare CTCs.

In this work, a novel flow cytometer NovoCyte® Quanteon™ was employed to measure and quantify spiked human tumor cells of colon carcinoma (SW620) in normal PBMCs or peripheral blood.

First, immunofluorescence labeled (EpCAM-APC antibody) SW620 cells (0–256 cells) were serially diluted in 1×106 PBMCs and tested directly on Quanteon. The data indicate that CTC detection using Quanteon has a sensitivity of 0.0001% (one CTC per million cells) and an excellent linear relationship between the number of cells detected and the predicted number added. Furthermore, SW620 cells (0–2000 cells) were serially diluted in 2mL normal whole blood, following the traditional isolation of PBMCs, magnetic EpCAM-positive selection, immunofluorescence labeling (CD45 FITC, EpCAM APC) and washing. The results show that the average recovery of CTCs is more than 75%.

Flow cytometry is a valuable tool for therapeutic monitoring and prognosis assessment. Detection of CTC is critical for a “liquid biopsy”. These experimental results demonstrate that the NovoCyte Quanteon flow cytometer can provide high specificity and accuracy of CTC detection through both direct identification and EpCAM affinity bead-based enrichment methods.
Inhibition of NK cell-MHCI interactions disrupts immune homeostasis by activation of the IFNγ/IL15 axis resulting in augmentation of anti-tumor immunity
Authors: Panda Abir Kumar , Shevach Ethan M
Published: January 2023

While TCR-MHC interactions are crucial for antigen recognition and induction of specific immune responses, all T cells have the potential to recognize “self” in the absence of foreign antigen. We have previously demonstrated that blockade of TCR-MHCII interactions in vivo resulted in a marked disturbance of immune homeostasis with an expansion of memory phenotype (MP) Treg, CD4+Foxp3−, and CD8+ T cells. To explore whether MHCI interactions also play a role in immune homeostasis, we treated unmanipulated mice with the pan-anti-H2 mAb, M1/42., for six days. Surprisingly, this treatment resulted in marked proliferation of NK cells, MP CD8+ T cells and to a lesser extent MP CD4+ T cells. All the effects of the anti-MHCI treatment were critically dependent on the presence of NK cells. Activated NK cells secreted high amounts of IFNγ which induced high levels of MHCI and MHCII expression on APC as well as IL-15 secretion by APC. IL-15 subsequently drove NK cell proliferation, and MP CD4+ and MP CD8+ T cell expansion. The global disruption of immune homeostasis induced by blockade of MHCI interactions created an environment that rendered the animals resistant to the growth of a transplanted tumor. Taken together, disruption of the interaction of specific lymphoid subpopulations with their MHCI targets in the steady state can paradoxically result in beneficial activation of immune responses against tumors. Blocking of MHCI interactions by anti-MHCI could also be useful to better understand immune responses against intracellular (viruses) and extracellular (bacteria) pathogens and the generation of long term memory responses.
TNF superfamily cytokines overcome immune evasion in medulloblastoma
Authors: Wechsler-Reya Robert , Garancher Alexandra , Suzuki Hiromichi , Haricharan Svasti , Masihi Meher Beigi , Rusert Jessica M. , Norris Paula S. , Carrette Florent , Romero Megan M. , Morrissy Sorana A. , Skowron Patryk , Cavalli Florence M.G. , Farooq Hamza , Ramaswamy Vijay , Jones Steven J.M. , Moore Richard A. , Mungall Andrew J. , Ma Yussanne , Thiessen Nina , Li Yisu , Morcavallo Alaide , Qi Lin , Henderson Jacob J. , Crawford John R. , Levy Michael L. , Olson James M. , Cho Yoon-Jae , Deshpande Ani , Li Xiao-Nan , Chesler Louis , Marra Marco A. , Becher Oren J. , Bradley Linda M. , Ware Carl F. , Taylor Michael D.
Published: January 2023

Many immunotherapies act by enhancing T cell killing of tumor cells. CD8+ cytotoxic T cells recognize antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. Here we show that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. Treatment with tumor necrosis factor (TNF) or lymphotoxin beta receptor agonist (LTβRag) rescues expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, TNF treatment prolongs survival and markedly augments the efficacy of the immune checkpoint inhibitor anti-PD-1. These studies identify p53 as a key regulator of immune evasion in vivo, and suggest that TNF could be used to enhance sensitivity of p53-mutant tumors to immunotherapy.
Identification and characterization of a unique KLRG1-expressing subset of CD4+FOXP3+ Tregs in Non-small cell lung cancer
Authors: Adeegbe Dennis O , Noyes David , Semidey-Hurtado Jon , Gamal Wael
Published: January 2023

It is well established that CD4+FOXP3+ T regulatory cells (Tregs) contribute to dampening anti-tumor responses. Despite our improved understanding of their role in cancer, there remains a knowledge gap with respect to the clonal composition and heterogeneity of tumor-infiltrating Tregs in solid cancers. We addressed this outstanding issue by conducting comprehensive phenotypic profiling of Tregs present in lung adenocarcinomas of a genetically engineered mouse model of non-small cell lung cancer (NSCLC) as well as those in resected tumors of NSCLC patients. Multi-parameter flow cytometric analysis revealed that unlike the peripheral tissues, the tumor harbors a distinct sub-population of Tregs that express the co-inhibitory receptor, KLRG1. Compared to their negative counterparts, the KLRG1+ Treg subset exhibited heightened expression of a number of Treg signature proteins as well as higher levels of activation and memory molecular markers suggesting that they are a highly activated and differentiated Treg pool that is recruited to, or induced in the tumor microenvironment. Consistent with this phenotype, these KLRG1+ Tregs were superior in their capacity to suppress T cell proliferation relative to the KLRG1-cells. Collectively, these findings demonstrate that the tumor microenvironment in non-small cell lung cancer harbors a unique Treg sub-population that is characterized by dominant expression of KLRG1, and which represents a clonal pool with the most potent inhibition of T cell responses. These studies highlight the installment of distinct Treg subsets in NSCLC that have implications for regulation of anti-tumor responses.
T-Cell Intrinsic Mechanisms of Resistance to PD-1 Checkpoint Blockade
Authors: Krogsgaard Michelle , Moogk Duane , Wang Lin , Li Kaito , Yuan Zhou , Weber Jeffrey , Osman Iman , Zhu Cheng
Published: January 2023

Although much clinical progress has been made in harnessing the immune system to recognize and target cancer, there is still a significant lack of an understanding of how tumors evade immune recognition and the mechanisms that drive tumor resistance to both T cell and checkpoint blockade immunotherapy. Our objective is to understand how tumor-mediated signaling through inhibitory receptors, including PD-1, combine to affect the process of T cell recognition of tumor antigen and activation signaling. Our goal is to understand the basis of resistance to PD-1 blockade and potentially identify new molecular targets to enable T cells to overcome dysfunction mediated by multiple inhibitory receptors. Biomembrane Force Probe(BFP) measurements show that that the activities of TCR-proximal signaling components affect T cell mechanosensing and sensitivity at the earliest stages of antigen recognition and are influenced by PD-1 and other inhibitory receptors via Shp-1/2 by targeting CD28 and Lckto directly suppress TCR-pMHC-CD8 binding. Phospho-proteomics and flow cytometry-based analysis of patient-derived T cells from PD-1 responders and non-responders identified additional mediators, signaling components and pathways associated with PD-1 checkpoint blockade resistance. CRISPR/Cas9 mediated genome editing was utilized to determine if resistance is mediated by the continued signaling of multiple IRs by perturbing IR signaling in mouse models of PD-1 blockade. Targeting these interactions and understanding the basis of resistance to PD-1 blockade would potentially allow identification of novel biomarkers of resistance or new molecular targets to enable T cells to overcome dysfunction during PD-1 checkpoint blockade.
The dark side of IL-1: role in prostate cancer progression
Authors: Udartseva Olga O , Gollnick Sandra O
Published: January 2023

IL-1 is a pro-inflammatory cytokine essential for host defense against pathogens, but its role in cancer progression remains understudied. Recently our lab identified the IL-1 pathway as a molecular mechanism responsible for myeloid-driven prostate cancer development. Here, we report that IL-1 signaling in tumor cells drives prostate cancer progression. To identify the contribution of IL-1 signaling in prostate cancer progression, we downregulated expression of IL1R1 (the only known functional receptor for IL-1α/β) using shRNA or Crisr/Cas9 in androgen-sensitive (LNCaP) and androgen-independent (PC-3M) prostate cancer cell lines. We found that IL-1α inhibited expression of androgen receptor in LNCaP while suppression of IL-1 signaling abrogated this effect. The expression of neuron-specific enolase which was shown to be a prognostic marker associated with poor survival outcome was significantly reduced in PC-3M cells lacking IL1R1. Downregulation of IL-1 signaling in cancer cells resulted in significant inhibition of prostate tumor growth while no effect on cell proliferation was detected. We established this phenomenon and found several possible mechanisms underlying it. We found that knocking down of IL1R1 in cancer cells resulted in suppression of mTOR and ACC signaling in tumors. TUNEL staining revealed increased number of apoptotic cells in shIL1R1-PC-3M tumors. Tumors lacking IL1R1 were characterized by impaired vascularization which was accompanied by reduced COX-2, VEGF-A and IL-8 expression. Taken together, our findings indicate that downregulation of IL-1 signaling modifies prostate cancer cell phenotype and suppresses tumor growth.
Immune checkpoint inhibitors reverse T-cell functional suppression in the bone marrow of a subset of AML patients
Authors: Lind Evan F , Lamble Adam J , Kosaka Yoko , Laderas Ted , Brady Lauren , Huang Fei , Druker Brian J , Tyner Jeffrey W , McWeeney Shannon
Published: January 2023

Acute Myeloid Leukemia is a blood cancer with poor prognosis despite decades of therapeutic development. Immune checkpoint inhibitors (ICIs) have been shown to be effective in many cancers, but their utility in AML has not been clearly established. To better understand the immune landscape of the AML microenvironment, we performed immunophenotyping on fresh bone marrow cells from AML patients using mass-spec based flow cytometry. In addition, we performed functional assays to evaluate T-cell proliferative capacity and determine whether AML bone marrow T cells can be modified by ICI treatment. Sixty two bone marrow samples from patients with AML were analyzed. We performed T-cell stimulation assays to measure proliferation and cytokine production in the presence and absence of ICIs. Samples were sub-categorized as being proliferators or non-proliferators based on the percent of T-cell division in response to TCR stimulation. Phenotypic analysis showed proliferation status correlated with a specific T-cell profile. Additionally, within the non-proliferator group, 70% overcame immune suppression and responded to at least one of the ICIs tested. In conclusion, we show that T-cell dysfunction is present in a subset of patients with AML. Nonetheless, this suppression can be reversed by ICIs in many cases, supporting the use of ICIs in AML therapy.
PD-1 checkpoint therapy promotes pro-survival phenotype in regulatory T cells in a model of claudin-low breast cancer
Authors: Vick Sarah C , Perou Charles M , Serody Jonathan S.
Published: January 2023

Results

We have previously shown in our claudin-low genetically engeneired mouse model (GEMM) that Tregs are increased in the tumor microenvironment (TME), and express high levels of PD-1. We hypothesized that one cause for the lack of activity of α-PD-1 therapy in GEMMs and patients with claudin-low tumors was the activation of PD-1 expressing Tregs in the TME. To evaluate this hypothesis, Tregs were isolated from claudin-low tumors and functionally evaluated ex vivo. In vitro, PD-1 blockade decreased the suppressive activity of Tregs at high Treg:Tnaive cell ratios, but no difference was found in suppression at physiologically relevant ratios. We compared the transcriptional profiles of Tregs isolated from tumor bearing mice with or without α-PD-1 therapy using RNA-Seq. There were 27 signifcantly differentially regulated genes in Tregs from mice treated with α-PD-1 compared to untreated. These genes were associated with survival pathways using IPA with JUN, FOS (P < 0.001) and BCL2 (P = 0.02) upregulated in Tregs exposed to α-PD-1 treatment. Based on these data, we hypothesized that α-PD-1 treatment on Tregs results in a pro-survival phenotype. We found in vitro that Tregs in the presence of α-PD-1 proliferated significantly more than untreated Tregs (P < 0.0001). Importantly, PD-1 blockade did not significantly alter the expression of immunosuppressive proteins such as CTLA-4, CD25, TGFβ, or Il-10 as measured by flow cytometry.

Conclusions

We found that PD-1 blockade increases proliferation of Tregs in the T11 model, while not significantly altering suppressive capabilities thus leading to enhanced immunosuppression in the TME. Activation of Tregs by α-PD-1 therapy may be responsible for hyperacute tumor flares post therapy.
CD39+CD8+ T cells exhibit a distinct phenotype among tumor-infiltrating tumor-antigenspecific CD8+ T cells
Authors: Lee Yongjoon , Park Junsik , Park Su-Hyung , Shin Eui-Cheol
Published: January 2023

Recent studies have spotlighted CD39 expression on tumor-infiltrating CD8+ T cells as an exhaustion marker (Cancer Research 2018, 1:78) and tumor antigen reactivity (Nature Communications 2018, 9:1). In the present study, we examined characteristics of CD39+CD8+ T cells among tumor-infiltrating lymphocytes (TILs) obtained from various types of tumors, including ovarian cancer, stomach cancer and hepatocellular carcinoma, to gain better understanding of CD39 expression on CD8+ T cells. To identify tumor antigen-specific CD8+ T cells, we used 11 HLA-A*A201 multimers specific for tumor associated antigens, including MAGE A1, NY-ESO-1, and AFP. CD39 expression of tumor antigen-specific CD8+ T cells was significantly higher in TILs than in peripheral blood lymphocytes. However, all of tumor-infiltrating tumor antigen-specific CD8+ T cells did not express CD39. As known previously (Nature 2018, 557:7706), tumor-infiltrating bystander CD8+ T cells, specific to viral epitopes, lack CD39 expression. Among tumor-infiltrating tumor antigen-specific CD8+ T cells, CD39+CD8+ T cells exhibited more severely exhausted features than CD39−CD8+ T cells. For example, the percentage of multiple immune checkpoint receptors-expressing cells was significantly higher in CD39+CD8+ T cells compared to CD39−CD8+ T cells. Moreover, higher proportion of T-betlow/Eomeshighcells and and lower expression of CD127 were observed in CD39+CD8+ T cells. In addition, CD39+CD8+ T cells expressed higher levels of tissue residency markers, such as CD103 and CD69. In conclusion, CD39+CD8+ T cells exhibit distinct phenotypes that represent severe exhaustion and terminal differentiation among tumor-infiltrating tumor-antigen-specific CD8+ T cells.
Viral infection induces the upregulation of MHC-1 expression in infected cancer cells
Authors: Kim Sang Hoon , Jung Yong Woo
Published: January 2023

Cancer cells can evade immune system by the downregulation of major histocompatibility complex-1 (MHC-1) molecules and upregulation of immunosuppressive ligands such as programmed death ligand 1 (PD-L1). In addition, cancer cells provide inhibitory cytokines so that the microenvironments of cancer maintain as an anti-inflammatory condition. However, it is not clear if cancer cells provide pro-inflammatory microenvironments to help clear viral infection, when they were infected with life-threatning viruses To test this question, we infected EL-4 cells, a lymphoma cell line, with lymphocytic choriomeningitis virus (LCMV) and examined if this infection causes the phenotypic change of cancer cells. On day 7 post infection, we observed that expressions of MHC-1 and PD-L1 molecules increased, while decreased PD-1 expression was found in virus-infected cancer cells. Since the overexpression of MHC-1 molecules was found on virus-infected cells, we further examined whether virus-specific CD8 T cells can recognize the antigen presented by these cancer cells and if these CD8 T cells can mount immune response. When LCMV-specific memory T cells were cultured with virus-infected EL-4 cells, we found that these CD8 T cells were able to produce IFN-γ and TNF-α, but the levels of these proteins were lower compared to positive controls. Altogether, these data suggest that cancer cells infected with viruses presented the viral antigens to CD8 T cells so that the delivery of viral antigen to cancer would be a novel therapeutic strategy.
Adrenergic stress impairs CD8+T-cell activation and effector function, limiting antitumor immune responses: an in vivo analysis
Authors: Qiao Guanxi , Chen Minhui , Bucsek Mark J , Mohammadpour Hemn , MacDonald Cameron R , Campbell Heather M , Hylander Bonnie L , Repasky Elizabeth A
Published: January 2023

CD8+T-cell activation requires a substantial up-regulation of both glycolysis and oxidative phosphorylation to support proliferation and immune effector functions. We have shown that chronic stress suppresses anti-tumor immunity and is associated with elevated levels of the sympathetic neurotransmitter norepinephrine (NE). NE signals through adrenergic receptors (ARs) present on all cells to coordinate a sympathetic stress response. We found that reducing or blocking AR signaling can significantly improve anti-tumor immune responses. Additionally, we discovered that triggering AR signaling during CD8+T-cell activation in vitro impairs metabolic reprogramming, primarily through the beta2-AR. We hypothesized that, in vivo, chronic stress would also impair metabolic reprogramming, leading to significant failure of intratumoral CD8+T-cell effector function. We found that the mitochondrial mass of CD8+T-cells isolated from B16-OVA tumors (melanoma) is decreased compared to CD8+T-cells from tumor draining lymph nodes; however, when tumor bearing WT mice are treated with propranolol, or in tumors grown in adrb2−/− mice, we found that tumor infiltrating CD8+T-cells have a higher mitochondrial mass. We also found that tumor infiltrating CD8+T-cells from mice treated with propranolol have higher CD28 expression, suggesting that adrenergic stress-induced signaling impairs metabolism of tumor infiltrating CD8+T-cells through a CD28-dependent pathway. Together, these data support the idea that in vivo, chronic stress inhibits the antitumor immune response through inhibition of CD8+T-cell metabolism.
Tumor-intrinsic PD-L1 regulates tumor initiating cell virulence and stemness genes, and TCF1+ stem-like T cells through Raptor in ovarian cancer, which correlates with survival in high grade serous ovarian cancer
Authors: Gupta Harshita B. , Murray Clare E , Deng June , Mohammad Tabrez AS , Zhang Xinyue , Wu Bogang , Clark Curtis A , Sareddy Gangadhara , Chen Yidong , Vadlamudi Ratna , Li Rong , Curiel Tyler J
Published: January 2023

We reported that tumor intrinsic PD-L1 promotes tumor initiating cell (TIC) virulence and mTORC1. Others have focused on PI3K/AKT/mTOR in promoting TIC, but specific downstream mTOR regulators of tumor stemness are unknown. We knocked down Raptor (mTORC1 component, Rptorlo) in ID8agg cells (murine ovarian cancer). Rptorlo cells had reduced mTORC1 with no change in PD-L1 in total cells or CD44+CD24+ TIC. %TIC was reduced in Rptorlo cell cultures, and Rptorlo TIC were functionally defective for self-renewal by tumorosphere formation. Rapamycin (mTORC1 inhibitor) reduced ID8agg Rptor, tumorospheres, and the stemness and virulence genes Oct4 and Nanog. Oct4 knockin to PD-L1lo ID8agg restored TIC numbers and spheres. Together, data confirm PD-L1 drives stemness through Oct4 by increasing mTORC1. Rptorlo TIC formed significantly smaller tumors vs. control TIC in immunodeficient NSG mice, confirming TIC intrinsic Rptor drives immune independent virulence. In wild type mice, Rptorlo vs. control TIC produced less ascites and smaller tumors with lower %TIC in vivo (but similar PD-L1), significantly more recently-activated CD44hiCD62LloCD8+ cells in ascites and draining lymph nodes (DLN). CCR2+CD8+ T cells and CXCR5+TCF1+PD-1+Tim3− stem-like T cells were also increased in Rptorlo DLN. Thus, Rptorlo TIC influence systemic and tumor-associated immunity. In high grade serous ovarian tumors (The Cancer Genome Atlas) high Rptor expression predicted a significantly higher cancer stem cell signature, and lower median survival in late stage patients. Our work establishes tumor intrinsic Rptor driven by PD-L1 plays a key role in regulating TIC and stem like T cells and can be a prognostic marker for late stage ovarian cancer patients.
Longitudinal Phenotyping Studies of MRL/MpJ-Fas
lpr/J and NZBWF1/J Mouse Lupus Models
Authors: Schile Andrew , French Richard , Serrano Jason , Leighton Krystal
Published: January 2023

MRL/MpJ-Faslpr/J (MRL-lpr) and NZBWF1/J mice develop autoimmune diseases resembling systemic lupus erythematosus, a disease affecting > 1 million Americans. MRL-lpr mice have a mutation (Faslpr) promoting survival of self-reactive lymphocytes and a strain background predisposed to autoimmunity. NZBWF1/J mice are a polygenic model with female disease bias. Cohorts of 40 mice/sex/strain were weighed and monitored weekly for clinical signs. Hematology, serum chemistry, and urinalysis was conducted on 10 mice/sex/strain every two weeks (MRL-lpr) or monthly (NZBWF1). Lymphoproliferation was greater in MRL-lpr females: white blood cell counts (×103/μL) increased from a median of 3.5 at 8 weeks to 9.2 at 18 weeks; median male counts increased from 3.2 to 6.1. At 38 weeks NZBWF1 WBC did not differ by sex, but red blood cells and platelets were significantly lower in females (14% & 55% reductions, respectively; P = 0.018 & P = 0.0001). MRL-lpr renal failure was progressive and greater in females: 9 of 10 females had urine albumin:creatinine ratios exceeding 100 mg/g by 17 weeks of age, while 2 of 10 males were in this range. Serum blood urea nitrogen increased by >3X in 5 of 7 NZBWF1 females that reached endpoint by 38 weeks; values did not change in MRL-lpr. The first overt signs of disease were cervical lymphadenopathy in MRL-lpr females and body weight loss in NZBWF1 females. More than 50% of female NZBWF1 mice had lost >10% of peak body weight by 38 weeks while all males were gaining weight. Median time to MRL-lpr study endpoint was 19 weeks for females and >24 weeks in males; mice generally maintained or gained weight throughout. All data will be available from the Mouse Phenome Database to serve as references for disease progression in these models.
IL-4 synergizes with low-dose IL-2 to restore systemic lupus erythematosus B cells at the resting naive status
Authors: Hsu Hui-Chen , Liu Shanrun , Gao Min , Wu Qi , Yang PingAr , Chatham W. Winn , Mountz John D.
Published: January 2023

We have recently identified that down-regulation of interleukin-4 receptor (IL-4R) is a novel hallmark of B-cell dysregulation in SLE patients. Single cell transcriptomics analysis revealed that elevation of IL4R in pre-switched B cells is the opposing signature of increased type I interferon stimulated genes (ISGs) and double negative B cells (TBX21, ITGAX, FCRL3, FCRL5, and ZEB2). Flow cytometry analysis further confirmed that down-regulation IL-4R and upregulation of intracellular IFN-β correlated with the loss of CD21+IgD+CD27− resting naïve (rNVA) B cells and accumulation of CD21−IgD+CD27− activated naïve (aNAV) and double negative CD21−IgD+CD27− B cells in SLE patients (n=47). Interestingly, exposure of SLE B cells to IL-4 prior to treatment with low-dose IL-2 (0.2 ng/mL) blocks the anti-Ig, IL-21, IFN-gamma, TLR7 and BAFF-stimulated development of T-bet+CD11c+IgD−CD27− DN2 B cells and CD27+CD38+ plasmablasts (PBs) and preserves B cells in the rNAV state. Such effect was diminished in the absence of IL-4 or when low-dose of IL-2 was substituted with high-dose of IL-2 (20 ng/mL). Stimulation of B cells with IL-4 promoted the induction of CD25 (IL-2RA) on B cells. Together, our results suggest a mechanism of action underlying the synergism between IL-4 and low-dose IL-2. Specifically, the IL-4R signaling pathways upregulate CD25 on B cells, thereby promoting formation of the high affinity IL-2R complexes required for low-dose IL-2 signaling that induces a regulatory signal that restores SLE B cells to the rNAV stage. Our results suggest that the process of development of autoreactive B cells in SLE can be directly measured in a clinical lab, and can be directly abrogated using cytokine therapy available for clinical use.
Lupus-like disease in B6.Nba2 mice shows a biphasic disease progression
Authors: Keller Emma J , Campbell Nicole , Davison Laura M , Patel Neeva , Jorgensen Trine N
Published: January 2023

Lupus is an autoimmune disease with a diverse pattern of symptoms affecting approximately 1.5 million Americans. Over the past decade, new biologics (f.ex. Belimumab, Anifrolumab) have emerged targeting cytokines and/or cells known to be pathogenic in lupus, however, response-rates among varied patient populations remain low, supporting the heterogeneity of the disease. B6.Nba2 mice exhibit a constitutive progressive lupus-like disease pattern driven by type I interferons (IFNα). Using this model, ablation of plasmacytoid dendritic cells (pDCs) is effective if initiated before, but not after, 4 months of age, suggesting a biphasic disease pattern with pDCs/IFNα playing a role during the early phase only. To investigate the second phase, we compared disease manifestations in 4 and 9 month old B6.Nba2 mice. As expected, IFNα-induced gene transcripts diminished after 4 months of age as did levels of IL-6; another product of pDCs, while we saw no changes in IL-21 levels. In contrast, splenic BAFF levels increased over time both at the transcriptional and protein level as did levels of Irf4 and Bcl6 transcripts, supporting an upregulation in specific B cell subsets including germinal center B cells and plasma cells in aging mice. These data indicates that pDCs and their products, IFNα and IL-6, play a critical role in early lupus like disease progression, while BAFF and B cells are involved in the later stage of the disease progression. Thus, treatment with different biologics may be needed at different time points of disease. We suggest that investigating patterns of gene expression combined with obtaining an inclusive patient history is necessary to determine the treatment approach needed for each individual lupus patient.
Interferon lambda promotes immune dysregulation and tissue inflammation in TLR7-induced lupus
Authors: Wang Xinghao , Goel Rishi R. , O’Neil Liam J. , Nakabo Shuichiro , Hasneen Kowser , Gupta Sarthak , Wigerblad Gustaf , Blanco Luz P. , Kopp Jeffrey B. , Morasso Maria I. , Kotenko Sergei V. , Yu Zu-Xi , Carmona-Rivera Carmelo , Kaplan Mariana J.
Published: January 2023

Type III interferon lambdas (IFN-λ) have recently been described as important mediators of immune responses at barrier surfaces. However, their role in autoimmune diseases such as systemic lupus erythematosus (SLE), a condition characterized by aberrant type I IFN signaling, remains to be determined. Here, we identify a non-redundant role for IFN-λ in immune dysregulation and tissue inflammation in a model of TLR7-induced lupus. IFN-λ protein is increased in murine lupus and IFN-λ receptor (Ifnlr1)-deficiency significantly reduces immune cell activation and associated organ damage in the skin and kidneys. Single-cell RNA sequencing in mouse and human immune cells revealed distinct and selective responses to this cytokine. Rather, IFN-λ activates keratinocytes and mesangial cells to produce chemokines that induce immune cell recruitment and drive tissue-specific inflammation. These data provide new insights into the immunobiology of SLE and identify type III IFNs as important factors for tissue-specific pathology in this disease.
B cell migration into skin limits local inflammation
Authors: Debes Gudrun , Aira Lazaro E , McGettigan Shannon E
Published: January 2023

Regulatory B cells (Bregs) limit inflammatory responses in various organs. While most studies suggest that Bregs exert their anti-inflammatory functions by virtue of IL-10 production in lymphoid tissues such as spleen and lymph nodes, we found that IL-10 producing B cells reside constitutively in skin and are preferentially recruited in inflammation in an α4β1 integrin-dependent manner. To address whether Bregs suppress inflammation at the effector site (i.e. inflamed skin), we employed mice with an inducible B-cell specific deletion of α4-integrin. Selectively blocking migration of B cells into the inflamed skin led to increased localized inflammation in mouse models of psoriasis and cutaneous hypersensitivity. The data support a model in which Bregs suppress inflammation not only in lymphoid tissues but also at effector sites.
Combined blockade of α4β7 and αEβ7 integrins leads to a greater reduction of gut proinflammatory T lymphocytes over either pathway alone
Authors: Dai Bingbing , Hackney Jason , Ichikawa Ryan , Elstrott Justin , Sun Kai-Hui , Modrusan Zora , Gogineni Alvin , McBride Jacqueline , Keir Mary E , Yi Tangsheng
Published: January 2023

Therapeutic approaches to treat inflammatory bowel diseases (IBD) include targeting integrins that mediate adhesion and migration of lymphocytes to the gastrointestinal (GI) tract. Etrolizumab is a humanized monoclonal antibody that selectively binds the β7 subunit of the heterodimeric integrins of α4β7 and αEβ7. The relative contribution of α4β7 and αEβ7 integrin family members to lymphocyte migration and retention in GI tractremains to be characterized. Here we applied single-cell technologies to healthy and IBD patient biopsies and found that αEβ7 is highly expressed in a subset of proinflammatory CD4+T cells and majority of cytotoxic intraepithelial CD8+T cells. In order to understand the role of αEβ7 in gut T cell accumulation, we used a photo-convertible reporter system to determine that concurrent inhibition of α4β7 and αEβ7 together led to a greater reduction of polyclonal or antigen specific T cell accumulation in the intestinal mucosa and epithelium compared to single blockade of either α4β7 or αEβ7. Further intra-vital two-photon microscopy and photo-specific labeling experiments revealed that blockade of αEβ7 reduces T cell interactions with basolateral epithelial surface, increases the migratory speed of activated T cells in the intestinal mucosa, and facilitates effector T cell egress from the intestinal mucosal through lymphatic vessels. Our data suggests co-blockade of α4β7 and αEβ7 together offers a superior inhibition of T cell accumulation in gastrointestinal tissues through a stepwise inhibition of T cell migration and subsequent tissue retention.
Full-length gasdermin-B (GSDMB) regulates epithelial adhesion and migration, and is increased in patients with IBD
Authors: Rana Nitish , Maywald Rebecca , Kondolf Hannah , Abbott Derek W , Heaney Jason , Ivanov Andrei , Pizarro Theresa
Published: January 2023

Resolution of inflammation at mucosal surfaces is a process critical for patients with IBD. GWAS revealed variant SNPs within the gene encoding gasdermin-B (GSDMB) that associates with an increased susceptibility to IBD and elevated GSDMB expression. The aim of this study was to determine the expression pattern and function significance of GSDMB in the pathogenesis of IBD. Primary intestinal biopsy and isolated intestinal epithelial specimens from IBD patients and non-IBD controls were evaluated for mRNA and protein expression. Using the human intestinal epithelial cell line, HT-29, CRISPR-cas9 knockout cells lacking GSDMB were generated, and used for in vitro XTT, cell-death and cell migration assays. Additionally, human epithelial cells transfected with GSDMB constructs and/or stimulated with methotrexate (MTX) were employed for relevant functional assays. We report for the first time a dramatic increase in full-length epithelial-specific GSDMB in IBD patients compared to healthy controls, which unlike other gasdermin family members, does not partake in program-mediated cell death. Additionally, we describe a functional decrease in in vitro proliferative activity, wound repair and an increase in focal adhesion proteins in epithelial cells lacking GSDMB, phenotypically rescued via transfection or MTX treatment. Importantly, GSDMB expression is decreased in IBD patients in remission, to that of levels of non-IBD controls. Taken together, our data suggests that increased epithelial-derived GSDMB during IBD is protective in function and promotes epithelial-specific adhesion and migration, providing rationale for the potential therapeutic use of GSDMB to optimize gut mucosal healing and restoration to homeostasis.
Spatiotemporal Dynamics of Regulatory T Cells and Th17 Cells in the Leptomeninges During Autoimmune Neuroinflammation
Authors: Othy Shivashankar , Jairaman Amit , Dynes Joseph , Dong Tobias X , Tune Cornelia , Yeromin Andriy V , Zavala Angel , Akunwafo Chijioke , Chen Fangyi , Parker Ian , Cahalan Michael D
Published: January 2023

Multiple sclerosis is a devastating autoimmune disease of the central nervous system (CNS) affecting more than 2.5 million people worldwide. Regulatory T cells (Tregs) and T-helper 17 (Th17) cells orchestrate anti- and pro-inflammatory responses that underlie autoimmune diseases. We used two-photon microscopy to elucidate spatial organization, interstitial motility, cellular interaction and signaling of endogenous cells within the spinal leptomeninges in an autoimmune model of neuro-inflammation (EAE). Th17 cells arrive early, distribute uniformly during peak, and decline in numbers during later stages of EAE. In contrast, Tregs arrive before the peak of EAE, co-localize with Th17 cells and persist during later stages. Th17 cells meander widely, whereas Tregs adopt confined, repetitive-scanning motility along the surface of antigen-presenting cells. Notably, Th17 cells exhibit robust Ca2+ signals at the disease onset that are suppressed in the presence of Tregs in the later phases, and Th17 cells co-existing with Tregs remain partially refractory to TCR-activation in-vitro. These findings indicate that interactions with Tregs impart long-lasting suppression of Ca2+ signaling on Th17 cells. Altogether, our results demonstrate choreography-based evidence for Treg control over encephalitogenic T cells to limit neuroinflammation during EAE.
IL-13 promotes precancerous epithelial cell changes during autoimmune gastritis
Authors: Noto Christine N , Bockerstett Kevin A , Jackson Nicholas M , Ford Eric L , DiPaolo Richard J
Published: January 2023

Individuals with chronic autoimmune diseases have an increased risk of cancer development in the affected organ. Cytokines released during chronic inflammation regulate tissue repair processes and can contribute to an increased risk of carcinogenesis. However, the identities and mechanisms by which cytokines regulate cancer development is poorly understood. In these studies, a TCR transgenic mouse model of autoimmune gastritis (AIG) was used to determine the cellular sources of IL-13 and the molecular mechanisms by which IL-13 promotes precancerous epithelial cell changes in a setting of chronic inflammation. Using this model, we identified IL-13 secreting CD4+ T cells, ILC2s, and mast cells present in the mucosa of chronically inflamed stomachs. The use of both murine and human 3D gastric organoid cultures established that IL-13 acted directly on the gastric epithelium and regulated epithelial cell survival, growth, and differentiation. Neutralizing IL-13 in mice with AIG prevented many of the precancerous cellular changes associated with gastric carcinogenesis. Finally, single-cell RNA sequencing of gastric epithelial cells with and without IL-13 receptors identified the transcriptional responses of mucosal epithelium to IL-13 during chronic autoimmunity. These studies have identified that tissue-specific IL-13 production during autoimmunity acts directly on the gastric epithelium to induce cellular changes associated with gastric cancer.
An engineered IL-2 partial agonist promotes CD8+ T cell stemness and anti-tumor efficacy
Authors: Mo Fei , Yu Zhiya , Li Peng , Oh Jangsuk , Spolski Rosanne , Zhao Liang , Glassman Caleb R. , Yamamoto Tori N. , Chen Yun , Golebiowski Filip M. , Hermans Dalton , Marjri Sonia S. , Picton Lora , Liao Wei , Ren Min , Zhuang Xiaoxuan , Mitra Suman , Lin Jian-Xin , Gattinoni Luca , Powell Jonathan D. , Restifo Nicholas P. , Garcia K. Christopher , Leonard Warren J
Published: January 2023

Adoptive cell transfer of antigen specific T cells represents a major advance in cancer immunotherapy, with robust clinical outcomes in a subset of patients. To achieve effective responses, both the number of transferred T cells and their cell differentiation state are critical determinants. T cells can be expanded with T cell receptor (TCR)-mediated stimulation and IL-2 but this can also lead to T cell differentiation into effector T cells, resulting in diminished therapeutic efficacy, whereas maintenance of a more stem-like prior to adoptive transfer is beneficial. Here, we show that an engineered IL-2 partial agonist generated on the super-IL-2 background promoted T-cell expansion without driving terminal differentiation. The partial agonist exhibited altered signaling and mediated distinctive downstream transcriptional, epigenetic, and metabolic programs. Moreover, it sustained expression of transcription factor TCF-1 (T cell transcription factor 1) and promoted mitochondrial fitness, facilitating the maintenance of a stem cell-like state. Accordingly, TCR transgenic and CAR-modified CD8+ T cells expanded with this engineered molecule displayed robust anti-tumor activity in vivo in established models of melanoma and acute lymphoblastic leukemia. Thus, tempering cytokine signaling with the IL-2 partial agonist provides a strategy for enhancing therapeutic efficacy by limiting exhaustion while preserving stemness. Moreover, our findings demonstrate the distinctive power of generation cytokine partial agonists with distinctive activities.
Training Next-Generation Immunologists on GuavaÒ MuseÒ Cell Analyzer Platform
Authors: Karp Jacob Alexander , Gillis Katherine , Salow Lisa , Dickey David , Cho Young , Madiraju Charitha
Published: January 2023

Immunology research in an academia setting is competitive and is especially challenging at any new professional program or new research laboratory. Faculty with start-up laboratories, welcome students into Biomedical Sciences research and in due course often seek for scientific collaborations. One such collaborative initiatives involved partnership between a non-profit academic research laboratory and Luminex, a biotechnology company that has a wide range of technological platforms including flow cytometers. This collaborative project is part of Research & Scholarship course (also called R&S course series) for PharmD students interested in Immunology research. The goal of the collaborative effort is to provide an opportunity for hands-on training of next generation immunologists on MuseÒ Cell Analyzer technology and proprietary kits/products designed for immunology and cell health analysis. R&S students are trained by their research faculty on an innovative microcapillary-based benchtop flow cytometer. Enrollment into Immunology-based research projects would entail students to train with a faculty mentor, conduct literature review on the assigned project, critically think about a research hypothesis, design methods, perform data analysis, arrive at a reasonably valid conclusion, and present a poster showcasing their results at an R&S poster day and/or other scientific conferences. Benchtop flow cytometry-based immunological and cell health assays relevant to R&S students’ research project and their execution using kits from Luminex are presented.