bluuebunny/crossref_metadata_2025 huggingface dataset:
Human B cell responses to cholera infection
Authors: Wrammert Jens , Kauffman Robert , Bhuiyan Taufiqur , Rashu Rasheduzzaman , Mayo-Smith Leslie , Khan Ashraful Islam , Chowdhury Fahima , Ryan Edward , Qadri Firdausi , Harris Jason
Published: January 2023

Vibrio cholerae causes an acute diarrheal disease estimated to lead to 3 to 5 million cases of cholera and over 100,000 deaths annually. Vaccine-induced immunity rapidly wanes and provides only partial protection. In contrast, natural infection induces 90–100 % protection for up to 10 years. However, it remains unknown why current vaccines are markedly less effective. Understanding mucosal humoral immunity and mechanisms regulating homing of immune cells to mucosal tissues in humans is of key importance, not only for V. cholerae infection, but for many other pathogens with a mucosal route of infection. To better understand the immune response induced by natural infection, we have characterized acute plasmablast responses in infected adults at the ICDDR, b in Dhaka, Bangladesh. To understand these potent responses in more detail we have generated a panel of 150 human monoclonal antibodies from plasmablasts isolated from six patients. These antibodies have provided a unique insight into the dynamics and variability of the functional characteristics of antibody responses to cholera, in terms of toxin neutralization, direct vibriocidal activity, the ability to induce bacterial agglutination or interfere with bacterial propagation. Furthermore, the fine specificity of these antibodies raise interesting questions about the longevity of B cell memory against bacterial polysaccharides. These studies provide unprecedented insight into heterogeneity of the acute plasmablasts responses to cholera, the ability to provide long-lived immunity after infection is resolved and the antigenic specificity of the BCR, at a single cell level. These findings may instruct future vaccine development for this important human pathogen.
Systemic dysregulation of cellular immune responses to H1N1 infection during pregnancy
Authors: Littauer Elizabeth Q , Esser E. Stein , Antao Olivia Q , Williams Dahnide T , Compans Richard W , Skountzou Ioanna
Published: January 2023

The 2009 H1N1 flu pandemic demonstrated that pregnant women infected with influenza were at risk for severe respiratory distress and premature-rupture-of-membranes (PROM), leading to high incidence of hospitalization, preterm births and small for gestation age (SGA) neonates. We utilize a syngeneic BALB/c pregnant mouse model which recapitulates clinical phenotypes shown during influenza infection of pregnant women. Pregnant mice sublethally infected (0.5xLD50) with pandemic H1N1 A/California/07/09 showed higher viral titers and delayed viral clearance relative to non-pregnant mice, and increased incidence of stillbirths and SGA offspring. Lymphocytes isolated at days 7 and 14 from lungs and spleens of infected pregnant and non-pregnant female mice were analyzed for H1N1 A/Ca/07/09 specific IL-4 and IFN-γ responses. Pregnancy delayed influenza-specific cytokine secretion at the site of infection, indicating systemic dysregulation of anti-viral responses. Lymphocytes from draining mediastinal lymph nodes, spleens and lungs were used to quantify activation of B cells in germinal centers (GC), maturation into antibody secreting cells and memory B cells, and mucosal homing to lung tissue following infection. Pregnancy decreased maturation of GC+ B cells in the spleen and migration of plasma cells from the spleen to the lungs. Infected pregnant mice generated equivalent serum influenza-specific neutralizing antibody titers and increased IgA antibody secreting cells (ASC) in the lungs 6 weeks post-infection relative to infected non-pregnant mice, indicating a potential role in pregnancy favoring the development of mucosal immunity in response to prolonged viral exposure.
TCR immune profiling reveals intrinsic clonal selection of tissue resident memory T cells in genital mucosa during human HSV-2 infection
Authors: Zhu Jia , Bedford Trevor , Williams Anthony , Laing Kerry , Jing Lichen , Li Alvason , Diem Kurt , Johnston Christine , wald Anna , Koelle David M , Corey Lawrence
Published: January 2023

Tissue resident memory (TRM) T cells persist in human genital mucosa long after healing of herpes simplex virus 2 (HSV-2) infection. These TRM T cells provide effective protection to rapidly contain reactivating viruses before clinical manifestation occurs, making them excellent candidates for developing novel approaches to controlling HSV2 reactivation. To understand the dynamics of TCR repertoire and the kinetics of T cell clones during disease resolution, sequential biopsies were obtained in three individuals at the time of active lesions and 2, 4, 8 weeks post-healing, and were subjected to high-throughput sequencing of the TCRβ chain hypervariable CDR3 region. Oligoclonal immune profiles were evidenced in all biopsy tissues with a few TCR sequences constituting 30–40% of the TCR repertoire. The usage pattern of various TCRVB genes was similar among tissues of active lesion and post healed biopsies of the same subject. However, the TCRVB usage differed dramatically between the TCR repertoire in tissues and those of PBMC derived HSV-2 specific T cells taken at the same time point within each individual. This difference was more extreme when evaluating the TCR sequences that selectively persisted in post-healing biopsies. The top ten predominant TCR sequences persisting in tissues during clinical quiescence 4–8 weeks post healing did not overlap with HSV-2 specific T cells isolated from PBMC. These data suggest a TCR intrinsic property might dictate the TRM repertoire, and that the antigen specificity of TRM might differ considerably from circulating HSV-2 specific T cells.
The establishment of a photoactivatable antigen system and its applications in B cell probing and B cell receptor sorting studies
Authors: Wang Jing , Liu Wanli
Published: January 2023

BCR and antigen recognition triggers a series of responses resulting in B cell activation and proliferation. It has been difficult to study the initiation of B cell activation due to its dynamic and fast nature. Here, we constructed a photo-activatable BCR ligand, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP) antigen that is biologically inert, and only becomes antigenic upon photo-activation with 405 nm photons. This photo-activatable antigen system provided a precisely controllable trigger point for B cell activation. This system in combination with live cell and single molecule imaging techniques by total internal reflection fluorescence microscopy has enabled us to illuminate the unidentified B cell probing termination behaviors and the precise BCR sorting mechanisms in the initiation of B cell activation. B cells in contact with the caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions, which is likely used by B cells to search for antigens in the proximal microenvironments. Such probing behaviors are cell intrinsic with strict dependences on F-actin remodeling but not tonic BCR signaling. B cell probing behaviors were terminated as quickly as 1–2 sec after photoactivation, suggesting probing termination response is sensitive and specific to BCR engagement. The termination of B cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. Lastly, single molecule imaging experiments demonstrated antigen-induced trapping of BCRs into the BCR microclusters as a fundamental mechanism for B cells to acquire antigens. Thus, a novel experimental approach was established for immune cell studies.
High Throughput Flow Screening Assays to Profile Cell-Mediated Killing
Authors: Luu Yen Kim , Liu Zhaoping , Duensing Thomas
Published: January 2023

While enhancing T-cell mediated killing of tumor cells is emerging as a successful therapeutic approach for a variety of cancers, improvements to these therapies are actively being sought. Traditional assays for monitoring cell-mediated killing are only capable of homogenous live/dead readouts for an entire sample. As an alternative platform for cell-mediated killing studies, IntelliCyt’s iQue Screener can identify multiple cell types in suspension and report multiple cell killing readouts in streamlined no wash assay formats. We demonstrate two example high throughput assays for cell-mediated killing using NK cells and chimeric antigen receptor (CAR) T-cells. Using the NK92 cell line as an effector cell and fluorescently encoded Jurkat cells as target cells, viability and Caspase 3 activation was determined for both Jurkat and NK92 cells in the same sample, and compounds that were generally cytotoxic to both cells could be identified. Specificity of the cell-mediated killing response was demonstrated using known signal transduction inhibitors including sunitinib, U73122, pp2, and wortmannin that would attenuate the NK cell killing activity, at a fixed target to effector cell ratio. In the CAR T-cell assay, efficacy of different CARs at targeting and killing a B-cell line (NALM-6) was profiled using multiplex readouts for cell health and secreted cytokines. Multiple cytokines including inflammatory markers and Granzyme B were quantified using bead-based ELISA on the same analysis platform. These application examples highlight the robustness and flexibility of the iQue Screener for performing multiplexed screening assays with cells and beads.
BIGG DATA: An accessible web portal for the analysis of immunologic repertoires.
Authors: Durrett Russell Eldon
Published: January 2023

The immune repertoire is an important resource for immunology research and for various clinical purposes. Technological advances over the past several years have led to an explosion of new techniques to capture the wealth of information contained in individuals’ Immunoglobulin and T-Cell Receptor repertoires, and large public datasets using these sequencing techniques have been made available in the last few years. However, a major challenge to understanding immune repertoire data is the high-level knowledge of computational infrastructure required to analyze these datasets, often requiring computationally-focused individuals to be recruited ad-hoc rather than simply being performed by the immunologists gathering the data. Herein, we have developed a simple, accessible web portal to aid in the analysis of these datasets. Our system allows users to bring their own raw datasets (in the form of FASTQ data), and utilize various popular, well-developed tools to derive VDJ-recombination annotations as well as to summarize statistical profiles of the full repertoire. This system provides an important, utilized resource that is employed by our lab for immunorepertoire analysis and is also available to the public in order to facilitate research in this area.
Computational analysis of epitope-specific T-cell repertoires.
Authors: Bradley Philip H , Fiore-Gartland Andrew , Hertz Tomer , Dash Pradyot , Thomas Paul
Published: January 2023

Experimental advances in flow cytometry, single-cell analysis, and high-throughput sequencing are leading to ever-increasing volumes of data on epitope-specific alpha/beta T-cell responses. Paired sequence data, in which the link between the alpha and beta chain sequences is retained, permit a more complete analysis than can be achieved with unpaired data: they allow reconstruction of molecular models for complete receptors, for example, and they enable a more precise assessment of TCR sharing across individuals. We are developing new algorithms for analyzing paired, epitope-specific TCR sequence data with the goal of investigating the mapping between TCR sequence and peptide-MHC recognition, using large, epitope-specific sequence datasets for model viral infections in mice. Our tools facilitate a variety of common repertoire analyses including landscape visualization (by multidimensional scaling or hierarchical clustering), CDR3 pattern detection, convergent evolution analysis, and detection of gene segment enrichment and alpha/beta pairing preferences. A null model for the rearrangement process allows us to assign significance scores to sequence motifs and patterns of TCR sharing across individuals. 3D structural data on TCR:pMHC complexes inform our clustering algorithms, and we use molecular modeling to aid in interpreting recurring TCR motifs. We are also pursuing machine learning approaches for selecting features predictive of binding. We believe these analysis and visualization tools represent an important first step toward developing predictive computational models of epitope recognition. We also expect that they may be useful to other researchers faced with large TCR repertoire datasets.
A scalable multiplex assay enabling assessment of T cell receptor specificity to hundreds of self- and pathogen-derived antigens
Authors: Klinger Mark , Taniguchi Ruth , Hu Joyce , Hayes Tim , Wittkop Tobias , Asbury Thomas , Moorhead Martin , Emerson Ryan , Sherwood Anna , Robins Harlan , Faham Malek
Published: January 2023

Monitoring antigen-specific T cells is critical for the study of immune responses and development of biomarkers and immunotherapeutics. We previously developed and validated a novel multiplex assay (MIRA, or Multiplexed Identification of T cell Receptor Antigen specificity) that combines conventional immune monitoring techniques and TCR repertoire sequencing to assess T cell specificity to large numbers of query antigens. MIRA is a sensitive assay enabling detection of antigen-specific TCR clonotypes well below the limit of detection of conventional immune monitoring assays including flow cytometry and ELISPOT. Here we report the results from a scaled-up version of the assay using 270 different query peptide antigens (159 self- and 111 pathogen-derived). We identified >500 TCR clonotypes at frequencies as low as 1 per million T cells that were specific to 41 query antigens from 6 healthy HLA-A*02-positive individuals. Most of the antigen-specific TCRs identified recognized one of 27 different peptides derived from a variety of pathogens including CMV, EBV, Flu, Rotavirus, HSV, mTB, WNV and HIV. A subset of antigen-specific TCRs recognized one of 14 different peptides derived from self including MART1, RCC, BCL-2, MAGE, STEAP1, KLK4, CAMEL and MOG. These data support the notion that escape and survival of self antigen-specific T cells occurs without causing overt autoimmunity in healthy individuals. We show here that MIRA can be used to assess TCR specificities to hundreds of query antigens simultaneously. The assay is highly scalable and can be easily modified to accommodate thousands of additional query antigens. This technology may be used to monitor T cell specificity to antigens relevant to infection, autoimmunity and cancer.
Profiling autoreactive IgA antibodies against 124 autoantigens in Systemic lupus erythematosus, systemic scleroderma and idiopathic inflammatory myositis
Authors: Li Quanzhen , Zhao Hongjun , Yan Mei , Zuo Xiaoxia
Published: January 2023

Systemic lupus erythematosus (SLE), systemic scleroderma (SSc) and idiopathic inflammatory myositis (IIM) are systemic autoimmune diseases characterized by existing of circulating autoantibodies (AutoAbs) to a broad range of self-antigens. In this study, we measured the serum levels of IgA AutoAbs against 124 self-antigens in a cohort of 128 SLE, 73 SSc, 75 IIM and 140 healthy controls (HC) using autoantigen microarrays. Our result indicated that IgA AutoAbs were highly prevalent in SLE and SSc compared with IIM and NC. 58.4% SLE and 41.7% SSc have 5 or more IgA AutoAbs compared with 16% in IIM and 14.1% in NC. 9 DNA specific IgA AutoAbs were significantly elevated in SLE including anti-dsDNA (54.7%), anti-ssDNA (51.1%), and anti-genomic DNA (46%). SSc showed significant increase of 32 IgA AutoAbs with the highest reactivity to Scl-70 (55.6%). 3 IgA AutoAbs significantly increased in IIM are Jo-1 (20%), Muscarinic receptor (8%) and B2-microglubulin (6.7%). Significant correlation was observed between IgA and IgG isotypes on most AutoAbs. 3 IgA autoAbs (anit-gDNA, anti-C1q and anti-dsDNA) positively correlated with SLEDAI Score, but only anti-C1q was significantly elevated in lupus nephritis. 14 anti-nuclear IgA AutoAbs negatively correlated with serum complement C3 and/or C4 levels.

This is the first study of large scale profiling of IgA AutoAbs in 3 systemic autoimmune diseases. Our study indicated that autoreactive IgA antibodies against different nuclear components are highly prevalent in SLE and SSC patients. IgA AutoAbs could be used as biomarkers for diagnosis and prognosis of systemic autoimmune diseases, and the molecular mechanisms underlining IgA AutoAbs production in systemic autoimmune diseases warrant further study
Bone marrow derived dendritic cells modified by lentiviral-mediated RelB shRNA possess tolerogenic phenotype and functions on lupus splenic lymphocytes
Authors: Mok Mo Yin , Wu Haijing , Lo Yi
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by high morbidity and mortality and remains challenging in treatment. Dendritic cells (DCs) have been shown to participate in the initiation and perpetuation of lupus pathogenesis. DCs that can induce tolerogenicity appear as potential cell-based therapy in this condition. In this study, we examined the in vitro tolerogenic properties of bone-marrow derived DCs (BMDCs) in the murine lupus setting. We used lentiviral transduction of RelB-silencing shRNA to modify expression of RelB, a key transcription factor regulating DC maturation, in BMDCs from MRL/MpJ mice. Tolerogenic properties of RelB-modified DCs were compared to scrambled control (SC)-modified DCs. RelB expression was found to be significantly reduced in RelB-modified DCs derived from MRL/MpJ mice, wild type of the same genetic background as MRL/lpr lupus-prone mice. These MRL/MpJ RelB-modified DCs displayed semi-mature phenotype with expression of lower levels of co-stimulatory molecules compared to SC-modified DCs. RelB-modified DCs were found to be low producer of IL-12p70, can induce hyporesponsiveness of splenic T cells from MRL/MpJ and lupus-prone MRL/lpr mice. Furthermore, they downregulated IFN-γ expression and induced IL-10 producing T cells in MRL/MpJ splenocytes, and attenuated IFN-γ and IL-17 expression in MRL/lpr splenic CD4+ lymphocytes. Splenocytes primed by RelB-modified DCs demonstrated antigen-specific suppressive effect on allogeneic splenocytes. In conclusions, RelB-silencing in DCs generates DCs of tolerogenic properties with immunomodulatory function and appears as potential option of cell-targeted therapy.
WX486, a novel inhibitor of BTK, demonstrated dose dependent inhibition of arthritis in mouse CIA and rat PG-PS models
Authors: Lin Jian , Chan Chichung , Wu Chengde , Shen Chunli , Han Shuhua , Li Xin , Guo Tingting , Chen Shuhui , Xiao Qiang , Huang Lu , Chen Hailiang , Tu Ronghua , Xu Yong , Wang Xuehai
Published: January 2023

Bruton’s Tyrosine Kinase (BTK) plays a crucial role in B cell differentiation and proliferation, making it an attractive target to treat inflammatory and autoimmune diseases. A small molecule BTK inhibitor, WX486, showed a high potency and selectivity, with an in vitro IC50 of 2.1 and 59nM in biochemical and cellular assays, respectively. In vivo anti-arthritis effect of WX486 was evaluated in two animal models, a mouse Collagen-Induced Arthritis (CIA) model and a rat peptidoglycan-polysaccharide (PG-PS) induced arthritis model. In both models, the administration of WX486 decreased ankle swelling and reduced morphologic pathology in a dose-dependent manner. In mouse CIA model, the inhibition of WX486 was 65.46%, 82.36%, 83.91% at 1, 3, 10mpk, respectively. In rat PG-PS model, WX486’s inhibition effect was 47.7%, 64.7%, 77.3% at 1, 3, 10mpk. In addition, WX486 at 3mpk demonstrated superior inhibition of arthritis over CC-292, a BTK inhibitor currently in phase II clinical trials for rheumatoid arthritis (RA), in both animal models.
Discovery of WX486 as a novel, potent, selective, and orally bioavailable irreversible inhibitor of BTK
Authors: Wu Chengde , Shen Chunli , Zhu Yuchuan , Hu Guoping , Lin Jian , Chen Shuhui , Xiao Qiang , Huang Lu , Chen Hailiang , Tu Ronghua , Xu Yong , Wang Xuehai
Published: January 2023

Bruton’s tyrosine kinase (BTK) has emerged as an attractive target for therapeutic intervention in human malignancies and autoimmune disorders, such as rheumatoid arthritis. Compounds were designed and evaluated in CADD based on published crystal structure. Selected compounds were synthesized and evaluated by BTK enzymatic assay. A chemical series where the central ring of ibrutinib was replaced by a bioisosteric motif showed strong enzyme inhibition activity. Further SAR of terminal aryl ring and Michael acceptor in terms of enzymatic activity, DMPK, hERG activity, and solubility was conducted. As a result, WX486 was identified as a potent (IC50=2.1 nM), selective (200~5000-fold over other 24 kinases), orally bioavailable (F=26.2%, 42.7%, 40.8% for mice, rats, and dogs, respectively), and soluble (solubility=266.40, 214.47, 209.01 μM at pH 2.5, 6.5, 7.4 respectively) preclinical candidate.
MyD88-dependent PKD1 is essential for development of polyarthritis spontaneously occurring in IL-1R antagonist-deficient mice.
Authors: Yi Ae-Kyung , Yoon Tae Won , Cho Hongsik , Park Jeoung-Eun , Hasty Karen A , Stuart John M
Published: January 2023

IL-1 receptor antagonist-deficient (IL-1rn−/−) mice develop spontaneous polyarthritis that mimics human rheumatoid arthritis. We investigated whether a Toll-like receptor (TLR)/IL-1R signaling adaptor MyD88 and its downstream protein kinase D1 (PKD1) play a role in the development and progression of arthritis in IL-1rn−/− mice. Serum cytokine levels, and macrophage accumulation, matrix metalloproteinase activities and cartilage damage in joints were significantly lowered in MyD88−/−IL-1rn−/− mice compared to MyD88+/+ IL-1rn−/− mice. Development of arthritis was completely inhibited in MyD88−/− IL-1rn−/− mice, indicating an absolute requirement for MyD88 in the development of arthritis in IL-1rn−/− mice. Because PKD1 is essential for proinflammatory responses mediated by MyD88-dependent TLRs, we further investigated the contribution of PKD1 to the development of arthritis in IL-1rn−/− mice. Suppression of PKD1 expression in vitro resulted in inhibition of TLR-mediated cytokine production in IL-1rn−/− macrophages. Daily treatment with a PKD inhibitor substantially reduced the incidence and severity of arthritis in IL-1rn−/− mice. In addition, deletion of the PKD1 gene in IL-1rn−/− mice using a tamoxifen-inducible system significantly delayed development of arthritis and reduced the incidence and severity of arthritis in IL-1rn−/− mice. Our findings demonstrated that MyD88 and PKD1 are necessary for development of arthritis in IL-1rn−/− mice. This also implies that PKD1 might be one of the key factors that modulate proinflammatory responses in rheumatoid arthritis, and may serve as a therapeutic target.
CancerNet: Molecular profiling of tumor microenvironment for biomarker discovery
Authors: Chenchik Alex , Makhanov Mikhail , Komarov Andrey , Sastry Sunitha , Frangou Costa
Published: January 2023

Human carcinomas consist of a complex mixture of neoplastic epithelial cells, endothelial cells, fibroblasts, myofibroblasts, and immune cells, which collectively form the tumor stroma. These cells are implicated in disease progression, metastasis and drug resistance. The clinical importance of tumor immune infiltrates has been an emerging area in triple negative breast cancer (TNBC) research where increased immune infiltrate predicts both response to chemotherapy and improved survival. Quantitative molecular profiles of tumor-associated normal cells may provide important insights into tumor biology and facilitate the discovery of new biomarkers and therapeutic targets. To this end, we have developed CancerNet, a comprehensive targeted RNA-Seq cancer immuno-panel that profiles ~2,000 genes and distinguishes 37 human hematopoietic cell phenotypes, (including naïve and memory B cells, seven T-cell types, dendritic cells, plasma cells, natural killer (NK) cells and myeloid subsets), genes involved in checkpoint blockade and immunotherapy biomarkers, immune cell activation and canonical immune pathway genes for both innate adaptive and humoral immune responses. In this study, we present an assay that characterizes the cellular composition of the immune/stromal/cancer cell tumor microenvironment and determines the activation status of infiltrating immune cells in primary tumor tissues from TNBC patients. Preliminary studies demonstrate the assay’s unparalleled specificity and sensitivity resulting in better detection of low abundance mRNA transcripts as well as an improved cost-effectiveness for high-throughput clinical Next-Gen applications.
Age effects on tumor B7-H1 and related immune therapies in an aggressive model of ovarian cancer.
Authors: Hurez Vincent , Padron Alvaro Souto , Drerup Justin Michael , Liu Aijie , Curiel Tyler J
Published: January 2023

Age is the greatest cancer risk factor, but contributions of aging to tumor intrinsic properties and immunotherapy responses are poorly understood as most preclinical studies are in young hosts. We assessed age effects on tumor properties, progression, immunity and response to immunotherapies in an aggressive ID8 ovarian cancer line (ID8agg) that is poorly immunogenic and fatal in ~5 weeks. Young (3 mos) and aged (24 mos) BL6 females were injected intraperitoneally with 4×106 syngeneic ID8agg cells. In young mice, ID8agg ascites had more regulatory T cells (Tregs) and myeloid suppressor cells (MDSCs). Young mice gained 30% weight from ascites ~30 days after ID8agg challenge and formed 1–2 large, discrete peritoneal tumor masses. Aged mice tended to lose weight although ascites volumes were comparable to young, suggesting cachexia, and formed multiple small peritoneal nodules. Aged ascites had more tumor cells, Tregs, monocytic MDSCs and PD-1+ T cells versus young. Strikingly, tumor cells and MDSCs in aged ascites upregulated the immune checkpoint molecule B7-H1 greater than in young ascites. The combination of depleting Tregs and MDSCs with denileukin diftitox (DD), an FDA-approved IL-2/diphtheria toxin + αGr-1 temporarily slowed tumor progression in aged mice with increased IFNγ+ CD8 T cells, consistent with our prior B16 data. Treg depletion with either DD or αCD25 alone, or plus αCTLA4 did not significantly prevent ID8agg progression in young or aged mice. In young hosts, αB7-H1 slowed tumor growth and increased survival, with both clinical outcomes potentiated by adding αCTLA4. αCTLA4 + αB7-H1 effects in aged hosts are under study. Thus, we identified major age effects on tumor progression, and immune and immunotherapy responses.
DEFINING THE IMMUNE MICROENVIRONMENT IN PATIENTS WITH ACUTE MYELOID LEUKEMIA
Authors: Lind Evan F , Lamble Adam , Kosaka Yoko
Published: January 2023

Introduction

Acute myeloid leukemia (AML) is the most common blood cancer in adults. Despite intensification of chemotherapeutic regimens, survival rates have plateaued. AML is therefore a candidate for novel immune therapeutics such as immune checkpoint inhibitors (ICIs). We aim to identify the relevant lymphoid and myeloid cell subsets present in the bone marrow of patients with AML, evaluating their differentiation and activation status. We are also performing functional assays to further define the status of T cells in the AML microenvironment.

Methods

Immunophenotyping was performed on mononuclear cells from fresh AML patient bone marrow using mass cytometry consisting of 5 panels of 35 antibodies each. Functional assays measured both the proliferative capacity and cytokine production profile of bone marrow T cells in the presence of various ICIs.

Results

Bone marrow samples from 17 patients with AML and 7 healthy donors have been collected and analyzed to date.

Conclusion

Our results show that the immune microenvironment is very heterogeneous between patients, with some differential responses to ICIs suggesting that certain subsets of AML may be potential candidates for immunotherapy.
CD49a and CD103 expression on tumor infiltrating lymphocytes in B16-OVA tumors depends on location and is correlated with a naive phenotype
Authors: Melssen Marit Martina , Rodriguez Anthony B. , Slingluff Craig L. , Engelhard Victor H
Published: January 2023

Tissue resident memory (TRM) cells are found in certain peripheral tissues long after resolution of infections that generate them. TRM formation is induced by TGFβ and has been associated with upregulation of CD49a and CD103. Interaction of CD49a and CD103 with their respective ligands is thought to allow long term persistence of TRM cells in peripheral tissues. In melanoma patients, increased expression of CD49a and CD103 has been observed on tumor infiltrating lymphocytes (TIL) compared to PBMCs. These phenotypic similarities suggest that a subset of TIL might function as TRM. However, the impact of continuous antigen presentation and/or differential TGFβ expression levels on this property of TIL is currently unknown. Here we demonstrate that, in B16-OVA melanoma tumors, CD49a and CD103 expression on CD8 TILs is upregulated compared to lymph node (LN) derived CD8 T cells from non-tumor bearing mice. Additionally, relative expression of CD49a and CD103 differs between subcutaneous and intraperitoneal tumors. The capacity of T cells to persist in tumors thus potentially changes based on tumor location, which may be dependent on TGFβ levels. In contrast to LN of non-tumor bearing mice, CD103 is expressed on 50–90% of LN CD8 T cells in tumor bearing mice. Furthermore, CD103+CD8 TILs have a more naïve phenotype, whereas CD103neg CD8 TILs mainly express memory/effector T cell markers. The opposite is observed when T cells are activated in vitro. These data suggest that CD103+CD8 T cells in tumors are largely due to a tumor induced systemic induction of CD103 on naïve T cells, rather than intratumoral TRM differentiation. The importance of CD103 in retaining naïve T cells for functional differentiation within the tumor remains to be established.
Hypofractionated radiotherapy treatment preserves immune function and improves tumor control vs. hyperfractionated treatment.
Authors: Barlow Margaret Lauren , Battaglia Nicholas , Gerber Scott A , Lord Edith M
Published: January 2023

Radiotherapy is one of the most effective means of treating solid tumors in cancer patients. The radiation acts in two ways to control tumor growth. First, by inducing cell death due to direct damage to the DNA, then by the dying tumor cells releasing cellular signals that boost antitumor immunity. Unfortunately, radiotherapy may also damage the healthy tissue surrounding the tumor, causing adverse side effects in patients. To limit this damage, radiotherapy in the clinic is generally delivered as multiple small doses (hyperfractionation) over the course of several weeks. However, in preclinical radiotherapy studies, radiation is often given as a single or a few high ablative doses (hypofractionation). We previously observed improved tumor growth control and an increased number of tumor infiltrating lymphocytes (TIL) in tumors given an ablative dose when compared to a hyperfractionated scheme. As TILs are radiosensitive, we hypothesize that the multiple doses of a hyperfractionated scheme may result in increased killing of the TILs, reducing the effectiveness of the anti-tumor immune response. To further investigate this, we compared the responses of the mouse Colon38 tumor model to a hypofractionated vs. a hyperfractionated scheme, using doses based on current clinical protocols. In vitro, the hypofractionation treatment was more effective at killing tumor cells and inducing the tumor cells to produce IL-1β, TNFα, IL-6 and IP-10. In vivo, the hypofractionated treatment also resulted in a significant delay in tumor outgrowth and improved survival rates when compared to the hyperfractionated treatment. Therefore, hypofractionated radiotherapy may increase immunity and be more beneficial in tumor control. Funded by NIH R01CA28332.
miR-30e* is overexpressed in prostate cancer and promotes NF-κB-mediated proliferation and chemotherapeutic resistance
Authors: Egan Shawn M , Gollnick Sandra O
Published: January 2023

Prostate cancer (CaP) is a critical problem for the aging male population. CaP is managed using a combination of prostatectomy, androgen ablation, chemotherapy and radiation. Subsets of patients develop androgen-insensitive metastatic CaP for which therapeutic options for are limited. Androgen-insensitive CaP clinically presents with constitutive NF-κB activation. NF-κB activation in the primary tumor is correlated with metastatic disease, biochemical relapse and drives tumor promoting chronic-inflammation. The NF-κB pathway is tightly regulated by many highly conserved mechanisms, the most well characterized being IκBα which sequesters NF-κB in the cytoplasm. The mechanisms that perpetuate constitutive NF-κB activation in CaP are largely unknown. microRNAs (miRs) translationally repress a number of genes involved with NF-κB activation. We report that miR-30e* is significantly overexpressed in multiple murine CaP models. miR-30e* functions to repress the translation of IκBα and drive NF-κB activation. The miR:30e* : IκBα axis drives CaP cell proliferation. miR:30e* drives cyclin D1 and subsequent phosphorylation of Rb which facilitates CaP cell proliferation. Inhibiting the miR:30e* : IκBα axis is sufficient to significantly increase the survival of mice bearing prostate tumors and reduce tumor burden. Additionally, inhibition of miR-30e* sensitizes CaP cells to the chemotherapeutic docetaxel. Elevated miR-30e* is a mechanism that CaP cells utilize to bypass critical NF-κB regulatory checkpoints and hijack this pathway. Targeting the miR-30e* : IκBα axis provides a novel opportunity to repair a critical checkpoint to NF-κB activation and open a window of therapeutic intervention.
Adoptive transfer of induced pluripotent stem cell-derived regenerated T cells mediates effective regression of large tumors and establishes immunological memory
Authors: Ito Fumito , Saito Hidehito
Published: January 2023

Current approaches to adoptive T cell therapy are limited by the difficulty of obtaining sufficient numbers of T cells against targeted antigens with effective in vivo characteristics. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSCs) that could provide an unlimited source of autologous T cells. However, therapeutic efficacy of iPSC-derived regenerated T cells remains to be elucidated. To this end, we established iPSCs from Pmel-1 T cell receptor (TCR) transgenic CD8+ T cells specific for the gp100 melanocyte differentiation antigen as a preclinical model of evaluating antitumor reactivity of regenerated antigen-specific T cells from iPSCs (Pmel-1 iPSCs). Pmel-1 iPSCs retained the same rearranged configuration as the original Pmel-1 CD8+ T cells, and differentiated into CD4+CD8+ double positive (DP) T cells that expressed CD3 and the desired TCR in vitro. Stimulation of iPSC-derived DP T cells with the cognate antigen in the presence of IL-7 and IL-15 followed by expansion with IL-2, IL-7 and IL-15 generated large numbers of less-differentiated CD8+ single positive T cells with antigen-specific potent cytokine production and cytolytic capacity. Furthermore, adoptively transferred iPSC-derived CD8+ T cells escaped immune rejection, mediated effective regression of large tumors, improved survival, and established antigen-specific immunological memory. These findings support the translational potential of iPSCs to provide an unlimited number of phenotypically defined, functional and expandable autologous antigen-specific T cells harboring characteristics necessary for in vivo effectiveness.
Imprime PGG, a yeast β-glucan PAMP elicits a coordinated immune response in combination with anti-PD1 antibody
Authors: Qiu Xiaohong , Chan Anissa S.H. , Jonas Adria Bykowski , Kangas Takashi , Ottoson Nadine R. , Graff Jeremy R. , Bose Nandita
Published: January 2023

Imprime PGG (Imprime), a b glucan PAMP is currently in clinical development in combination with tumor-targeting antibodies, anti-angiogenic antibodies and immune checkpoint inhibitors. Randomized Phase 2 clinical trials of Imprime in the 1st-line treatment of stage IV non-small cell lung cancer have shown promising efficacy in terms of both objective tumor response and survival. As a PAMP, Imprime is readily recognized by innate immune cells, triggering immune responses including repolarization of M2 macrophages and dendritic cell (DC) maturation. Earlier results have shown that both the M2 macrophages and DC derived from Imprime-treated monocytes have higher expression of PD-L1 and CD86. Since PD-L1 expression, a marker for adaptive immune resistance, and CD86, a co-stimulatory marker for T cell activation could potentiate the activity of anti-PD1 antibody, we hypothesized that the combination will show synergistic T cell stimulatory activity. Ex- vivo treatment of T cells with anti-PD1 antibody, nivolumab showed increased proliferation in response to CD3/CD28 stimulation. Coculture of these T cells with Imprime-treated M2s or DCs drove further T cell expansion and increased production of several cytokines including IFN-γ, IL-2, TNF-α, GM-CSF, and IP-10. We next evaluated the anti-tumor efficacy of Imprime and murine anti-PD1 in a syngeneic tumor model. In the CT-26 syngeneic model, the median tumor volume in the anti-PD1 alone treatment group was 936 mm3, while it was only 172 mm3 in the Imprime and anti-PD1 combination group. Collectively, these data suggest that the combination of Imprime and checkpoint inhibitors holds a lot of promise for cancer immunotherapy.
Shortened T cell culture with IL-7 and IL-15 provides the most potent chimeric antigen receptor (CAR)-modified T cells for adoptive immunotherapy.
Authors: Ghassemi Saba , Bedoya Felipe , Nunez-Cruz Selene , June Carl , Melenhorst Jos , Milone Michael
Published: January 2023

Adoptive T cell immunotherapy involves the isolation, ex vivo expansion and reinfusion of T cells, and is dependent on T cell engraftment and persistence for efficacy. IL-2 is the most commonly used cytokine for ex vivo T cell culture; however, there is renewed interest in IL-7 and IL-15 due to their ability to enhance the survival and proliferation of stem cell memory (Tscm) T cells. Peripheral blood (PB) contains substantial numbers of Tscm, central memory (Tcm) and effector subsets. During ex vivo culture, PB T cells differentiate into cells with a predominantly CD45RO+, CD27−, CCR7− effector phenotype. Exogenous IL-7/15 delays this transition in phenotype and preserves a greater proportion of Tscm and Tcm cells. We hypothesize that limited ex vivo T cell culture in the presence of IL-7/15 rather than IL-2 will enhance engraftment and persistence of T cells in vivo. We show that T cells can be harvested from ex vivo cultures as early as day 3 (d3) following activation. T cells expressing a chimeric antigen receptor targeting CD19 (CART-19) show potent yet specific cytotoxicity in vitro. We investigated the therapeutic potential of cells harvested at d3 versus later time points using a Nalm-6 leukemic cell xenograft mouse model. We demonstrate that d3 CART-19 cells show potent anti-leukemic activity compared to day 5 or day 9 cells. Comparing CART19 cells cultured in IL-2 or IL-7/15 for 3 or 9 days, we show that mice treated with d3 cells cultured in IL-7/15 exhibit the greatest anti-leukemic efficacy at a 10-fold lower dose compared with day 9 cells. In summary, we show that limiting T cell culture ex vivo to the minimum required for lentiviral transduction, in the presence of IL-7/15, provides the most efficacious T cells for adoptive immunotherapy.
High-Content CD14+ Cells in Apheresis Products negatively affects the Transduction and Expansion of CAR T cells during Large-scale cGMP Manufacturing
Authors: Riviere Isabelle , Qu Jinrong , Stefanski Jolanta , Borquez-Ojeda Oriana , Hack Anniesha , Wang Xiuyan
Published: January 2023

Adoptive transfer of chimeric antigen receptor (CAR) engineered T cells is a promising strategy to treat patients with cancer. Apheresis products are commonly used as starting material for large-scale T cell selection using CD3/28 magnetic beads. At the time of collection, the composition in various cell lineages is highly variable. We have encountered manufacturing challenges with apheresis products containing high proportion of CD14+ cells. As an illustration, we investigated the transduction and expansion of T cells using an apheresis product derived from a patient with ALL and containing 70% CD14+ cells. 350 × 106 CD3+ cells were selected from a cryopreserved apheresis product using CD3/28 magnetic beads. A short adherence procedure decreased the CD14+ cell composition from 70% to 45%. On day 3, 350×106 vs 20 ×106 total cells were recovered from each group without and with the depletion step, respectively. Selected cells from both groups were transduced using the same dilution of vector stocks at a cell concentration of either 0.4×106/mL or 0.2×106/mL in tissue culture bags. We observed a drastic difference in transduction efficiency between the group without depletion (4.5%) and the group in which the CD14+ cells were depleted (38%). Cells from the depleted group expanded 350 fold from day 3 to day 10 (100% CD3+). Our results suggest that high CD14+ cell content poses a manufacturing challenge when the selection of the CD3+ cells in the apheresis product is performed with CD3/CD28 magnetic beads. The CD14+ cell threshold for successful manufacturing without depletion will be discussed and additional samples will be analyzed.
Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for TCR and CAR based Adoptive Immunotherapy of Cancer
Authors: Sukumar Madhusudhanan , Restifo Nicholas
Published: January 2023

Long-term survival and anti-tumor immunity critically depends on their metabolic fitness but approaches to select metabolically robust T cells for T cell based immunotherapy remains unknown. We utilized a lipophilic cationic dye (tetramethylrhodamine methyl ester) to identify and isolate metabolically robust T cells based on mitochondrial membrane potential (ΔΨm). Cells with lower membrane-potential (low ΔΨm) had a molecular profile of memory precursors and displayed an enhanced ability to enter the memory pool as compared to cells displaying higher mitochondrial potential (high ΔΨm) characteristic of short lived effectors. Interestingly, we also found that multiple distinct negative inhibitory receptors such as PD1, CTLA4, BTLA, LAG3 and TIM3 were enriched in the high ΔΨm subset compared to the low-ΔΨm subset. Comprehensive metabolomics profiling demonstrated global features of metabolic fitness (increased lipid oxidation) in low ΔΨm sorted CD8+ T cells. Transfer of low ΔΨm T cells was associated with superior long-term in vivo persistence as evidenced by 100 fold increase in the frequency of T cells 300 days after adoptive transfer, augmented autoimmunity and an enhanced capacity to eradicate established cancer compared with high ΔΨm cells. Mechanistically, we found that the IL2 driven mTOR activation caused increased mitochondrial oxygen consumption and accumulation of higher level of ROS. Surprisingly, use of Ψm to enrich for cells with superior metabolic features was observed within central memory, effector (Tc17, Th1, and Th17) T cells and long term hematopoietic stem cells (LT HSC). This novel metabolism based approach may be applicable to therapies involving transfer of lymphocytes for treatment of advanced cancer.
Models of TCRαβ/CD19-depleted hematopoietic stem cell transplantation
Authors: Baiu Dana C , Otto Mario
Published: January 2023

Accelerating the reconstitution of protective immune T cell populations and immune function, while avoiding graft-versus-host disease (GVHD), is crucial for improving clinical outcomes following hematopoietic stem cell (HSC) transplantation. We developed and validated a clinical HSC graft engineering technique that selectively depletes αβ TCR T cells and CD19+ B cells from the graft, thereby minimizing GVHD and lymphoproliferative disease while maintaining HSC and important immunoprotective cell populations in the graft. The enrollment of the first patients in a University of Wisconsin conducted phase I clinical trial using this TCRαβ/CD19-depletion method in haploidentical HSC transplantation is to begin in the current year. In parallel, we developed preclinical models to explore the post-transplant HSC persistence and differentiation, and to evaluate the role of accompanying cell populations in facilitating HSC engraftment and immune recovery. We evaluated the efficiency of both CliniMACS and AutoMACS αβ TCR T cell depletion procedures by flow cytometry. Threshold numbers of contaminating αβ TCR T cells capable of inducing acute GVHD were determined after adoptive cell transfer in immunodeficient NOD.Scid.gamma mice. In vivo post-transplant persistence of αβ TCR and of γδ TCR T cells, NK cells and CD34+ engraftment were determined by flow cytometry at early (1–4 week) and late (5–12 week) time points. In addition to monitoring the post-transplant animal health status as a measure of graft alloreactivity/xenoreactivity, T cell activation markers (CD197, CD45RA) were assessed by flow cytometry. The murine models we developed will be instrumental in determining the effect of drug therapies on T cell reconstitution.
Temporal changes in tumor-associated immune cell populations in subcutaneous Tu-2449SC tumors transduced with Toca 511 following treatment with 5-fluorocytosine
Authors: Robbins Joan , Mitchell Leah , Yagiz Kader , Espinoza Fernando Lopez , Mendoza Daniel , Jolly Douglas J
Published: January 2023

Toca 511 (vocimagene amiretrorepvec), a gamma retroviral replicating vector encoding cytosine deaminase, with 5-fluorocytosine (5-FC), kills tumor by local production of 5-fluorouracil (5-FU), and induces local and systemic immunotherapeutic response resulting in long-term survival after cessation of 5-FC. This work identified the immune cell populations that change over time following administration of 5-FC to mice bearing Tu-2449SC tumors expressing cytosine deaminase.

Tu-2449SC cells (2% pretransduced with Toca 511) were injected subcutaneously in B6C3F1 mice. 5-FC or PBS treatment was initiated once tumors were palpable. Tumor burden was significantly reduced in mice that received 5-FC vs. PBS control. At 3, 6, 9, and 14 days after 5-FC start, spleens and tumors were harvested for immunophenotyping. 3 days after treatment initiation, B cells and CD4+ T cells in the tumor were significantly reduced with 5-FC. Of the CD4+ T cells that remained in 5-FC treated tumors, a greater number expressed markers of activation (41BB, CD40L, and PD-1), were defined as effector T cells, and fewer were naïve.

At day 6 post 5-FC initiation, CD4+ T cell percentages were no longer reduced with 5-FC vs. PBS. Tumor associated macrophages (TAM), myeloid derived suppressor cells (MDSC), and monocyte populations were significantly reduced in tumors 6 days after 5-FC start. Myeloid cell depletion correlates with previous work with systemic 5-FU.

At 14 days post 5-FC, TAM and MDSC remained reduced in tumors of treated animals, and both CD4+ and CD8+ T cells were significantly increased.

Toca 511+5-FC treatment results in reduced tumor burden and creates a tumor microenvironment that is more permissive to immune activation and anti-tumor immune response.