bluuebunny/crossref_metadata_2025 huggingface dataset:
Zinc mediates the interplay between Th1/Treg differentiation and regulates anti-tumor immunity
Authors: Sugandha N , Rizvi Zaigham Abbas , Dalal Rajdeep , Adhikari Neeta , Awasthi Amit
Published: August 2023

Dietary supplements play an important role in determining the health and immunity. Zn supplement has been used for the dietary chemoprevention of cancer. However, some of the studies say that Zn is important for cell proliferation and cells in the Zn deficient condition arrested in the S-phase of cell cycle. Dietary Zn deficiency is known to inhibit several types of animal tumors. Cancer cells have upregulated Zn importers and increased Zn levels and Zn deficiency induces cell death via apoptosis. As Zn supplementation is widely used a detailed study analysing the effects of Zn on tumor progression has become the necessity of this hour.

In this study we have identified that providing higher doses of Zn supplement for longer duration during tumor development results in increased tumor burden. Zn supplementation during tumor development alters mainly CD4 T cell subtypes by suppressing T helper 1 (Th1) cell population and enhancing T regulatory (Treg) cell population. Zn supplementation significantly increases PD1 immune checkpoints on CD4 T cells, CD8 T cells and γδT cells. The degranulation marker CD107a or LAMP1 expression on NK cells and CD8 T cells decreased indicating the decrease in cytotoxicity among these cells by Zn supplementation.

Our findings open excellent therapeutic avenues for targeting cancer. Firstly use of Zn chelators as a tumor therapeutic agent. Zn chelators can be combined with α-PD1 mAb checkpoint therapy to overcome the drawbacks of immune checkpoint therapy and efficient treatment outcome. Secondly trace element analysis can become the forefront of cancer immunotherapy, as our study along with many other reports prove that the level of trace elements determines the tumor development and therapeutic outcome.

Supported by grants from Department of Bio-Technology (DBT), Ministry of Science and Technology, Govt. of India.
mRNA-HPV Therapeutic Vaccine Encoding E6 and E7 Improves Therapeutic Potential in HPV-mediated Cancer
Authors: Lee Seonghyun , Yoon Hyunho , Hong Seol Hee , Bang Eun-Kyung , Kwon Sung Pil , Kwak Hye Won , Park Hyo-Jung , Lee Jisun , Park Hyeong-Jun , Bae Seo-Hyeon , Youn Hyewon , Keum Gyochang , Lee Shee Eun , Nam Jae-Hwan
Published: August 2023

The viral E6 and E7 oncoproteins of human papillomavirus (HPV) are strongly associated with cervical cancer. We developed and assessed the efficacy of an mRNA-HPV therapeutic vaccine (mHTV), containing the E6 and E7 proteins, in C57BL/J mice. mHTV generated robust T cell-mediated immune responses and induced short-lived effector and E7 tetramer-specific T cells. mHTV immunization significantly suppressed tumor growth in mice implanted with TC-1 tumor cells and improved overall survival. The re-transplantation of tumor cells into mice that survived the primary tumor challenge after primary vaccination led to the complete inhibition of tumor progression in all mHTV-treated mice. This showed that mHTV can effectively suppress cancer recurrence. mHTV also increased the number of tumor-infiltrating lymphocytes in the tumor tissues. When mice were implanted with TC-1 tumor cells after mHTV vaccination, most of the immunized mice were protected from the tumor challenge. In conclusion, mHTV showed considerable potential as not only a therapeutic vaccine, by efficiently killing cancer cells, but also as a prophylactic vaccine by enhancing T cell-mediated immune responses.

The Brain Korea 21 Plus Program, Partially supported. The Ministry of Food and Drug Safety grant (grant number 22213MFDS421) The National Research Foundation of Korea (NRF) grant (grant number NRF-2021M3E5E3080558)
T-cell and B-cell receptor repertoire profiling for biomarker discovery
Authors: Chenchik Alex , Makhanov Mikhail , Liu Tianbing , Hu Dongfang , Diehl Paul , Kobzik Lester
Published: August 2023

T-cell receptor (TCR) and B-cell receptor (BCR) repertoire profiling, also referred to as adaptive immune receptor repertoire (AIRR) profiling, holds great potential for the understanding of disease mechanisms and for the development of new treatments in infectious disease, autoimmunity and immuno-oncology. This potential could be greatly improved by combining information about receptor clonotypes with immunophenotypes of T- and B-cells. We developed a new technology for combined profiling of all human TCR and BCR variable regions and phenotypic characterization of immune cells in the same workflow. The TCR and BCR immunophenotyping method involves RT-PCR amplification and sequencing of the CDR3 regions of the TCR and BCR genes, as well as determining the expression levels of the most informative T- and B-cell phenotyping genes. Results show that this method allows for comprehensive profiling of all seven TCR and BCR chains from a single sample, in a highly reproducible manner, directly from micro-samples including cancer tissue, whole blood, sorted cells and more. Bioinformatic analysis of the next-generation sequencing (NGS) data allows profiling of the TCR and BCR clonotypes and identifcation of major immune cell subtypes and their activation status. Preliminary data from rheumatoid arthritis blood samples and others will be presented.
Development of a liquid tumor potency assay for evaluation of immune cell-mediated cytotoxicity in vitro
Authors: Califano Danielle , Streeter Ben , Sullivan Denise , Passaro Austin , Chvatal Stacie , Millard Daniel
Published: August 2023

The development of immunotherapies relies on the use of in vitro potency assays—which are key for understanding complex interactions between immune (effector) cells and cancer (target) cells—to evaluate the function, specificity, and sensitivity of a product. A variety of in vitro assays are used to characterize the proliferation, cytokine release, and cell-mediated cytotoxicity of engineered immune cells, such as chimeric antigen receptor (CAR) T cells, with each representing an important aspect driving clinical efficacy of the cellular product. Previous work has shown that impedance-based potency assays provide a real-time, label-free measure of effector cell-mediated cytotoxicity for adherent target cells. Here, we describe an in vitro potency assay that quantifies effector cell-mediated cytotoxicity of liquid tumor target cells. First, the wells were coated with antibody to tether the liquid tumor target cells (Raji = anti-CD40, K562 = anti-CD71, Daudi = anti-CD40). Tethering the liquid tumor target cells significantly increased the impedance measurement, as compared to untethered controls. CD19-specific CAR T effector cells were added 24 hours after the target cells and co-cultured with the target cells across a range of effector to target ratios. Cytolysis of the target cells was calculated by comparing the impedance in treated wells to no treatment control wells. As expected, the CD19-specific CAR T cells produced significantly higher cytolysis in the Daudi (54.3%) and Raji (70.4%) cells, which express CD19, as compared to the K562 cells (30.5%). These results support a real-time, label-free potency assay with liquid tumor target cells.
Dual targeting of mast cells and TSLP with a bispecific antibody
Authors: Alvarado Diego , Vitale Laura , Murphy Michael B , Wasiuk Anna , Widger Jenifer , Crocker Andrea , Patterson Colleen , Mills-Chen Laura , O’Neill Thomas , Baronas April R , McManus Kelly M , Boyer James M , Montanari Noe Rico , Lillquist Jay , Malenchek Linda , Weidlick Jeff , Goldstein Joel , Thomas Lawrence J , Marsh Henry C , Keler Tibor
Published: August 2023

Rationale

Reduction of mast cells (MCs) through stem cell factor (SCF) neutralization combined with inhibition of the alarmin TSLP in a single molecule may result in broader efficacy in inflammatory disorders.

Methods

Monoclonal antibodies (mAb) separately targeting SCF and TSLP were generated from immunized mice. A potent neutralizing antibody against each target was selected and humanized. Bispecific tetravalent antibodies (bsAbs) with specificity towards SCF and TSLP were generated. Candidate bsAbs were tested in vitro for neutralizing activity and evaluated in pilot non-human primates (NHP) studies.

Results

We discovered a novel anti-SCF neutralizing mAb (SCF-12) that potently inhibits SCF-dependent KIT phosphorylation and SCF-enhanced degranulation of primary human MCs. Separately, a novel anti-TSLP mAb (1D10) inhibits TSLP receptor binding and TSLP-dependent TARC release from human monocyte-derived DCs. bsAbs combining humanized SCF-12 and 1D10 with good expression, biophysical profile and robust neutralizing activity against each target were generated. Following demonstration of MC depletion in NHPs with SCF-12, we conducted a pilot NHP study with two lead candidate bsAbs. Pharmacokinetic, pharmacodynamic and tolerability data from this study will be presented.

Conclusions

SCF x TSLP bsAbs were successfully generated from antibodies that neutralize soluble SCF, leading to MC reduction in NHP, and TSLP, a key alarmin implicated in inflammatory and autoimmune disorders. Combined inhibition of these two pathways using a single molecule may be of broad utility in multiple inflammatory disorders.

Celldex Therapeutics
Analysis of T Cell Immunity Generated by Takeda’s Tetravalent Dengue Vaccine
Authors: Omidi Nazanin , Victor Kaitlin , Gray Lauren , Nadeem Iqra , Bolton Jessica , Robinson Tanisha , Hatch Kristin , Sharma Mayuri , Mandaric Sanja , Tricou Vianney , Currier Jeffrey , Friberg Heather
Published: August 2023

Dengue is a viral disease transmitted to humans through the bite of infected mosquitoes and is caused by dengue virus (DENV), which has four distinct serotypes, DENV1 to DENV4. Severe dengue is a leading cause of serious illness and death in some Asian and Latin American countries. The Walter Reed Army Institute of Research (WRAIR) has been working to investigate the cell mediated immunogenicity of Takeda’s live attenuated virus tetravalent dengue vaccine (TAK-003). Previously, we have used ELISPOT and Flow-ICS assays to show that TAK-003 induces a robust IFN-γ producing DENV-specific T cell response that is durable to at least 6 months post-vaccination. In addition, this response is mediated by both CD8+ and CD4+ T cells that produce IFN-γ and/or TNFα and IL-2. Using samples from a phase II study, DEN-313 (NCT02948829) conducted in healthy children aged 4 to 16 years in dengue endemic locations, herewith we extend the findings on TAK-003 induced DENV-specific T cell responses to: (1) present data on durability of the CMI response through 3 years post-vaccination; (2) verify the serotype coverage of the T cell response; (3) assess multifunctional capacity of the durable memory T cells, and (4) describe the phenotypic changes that occur among these cells based upon central memory (CD45RA−/CCR7+), effector memory (CD45RA−/CCR7−) and effector memory RA phenotyping (CD45RA+/CCR7−). Our data describe the frequency, functionality, and memory phenotype of T cells induced by TAK-003 up to 3 years post vaccination.

Finding received through corporative research and development agreement with Takeda vaccine company.
Double Negative B cells are decreased in patients with Idiopathic Inflammatory Myopathies
Authors: Reyes Raul Fernando , Torres-Ruiz Jiram , Gómez-Martin Diana , Maravillas-Montero José Luis
Published: August 2023

Background

Idiopathic inflammatory myopathies (IIM) are systemic autoimmune diseases characterized by skeletal muscle inflammation, frequently accompanied by extra muscular features associated with specific autoantibodies. The detailed pathogenesis of IIM remains unclear, but evidence suggests autoreactive B cells may play an essential role in this process. However, little is known about changes in peripheral blood B cell subsets in adult IIM patients.

Objective

This study aims to determine the frequency of B cell subsets among patients with IIM and their clinical correlation with disease activity.

Methods

Using a multiparametric flow cytometric approach, we determined the frequencies of B cell subsets in a cohort of 36 patients with IIM and correlated their values with clinical and laboratory data.

Results

DN B cells (CD27− IgD−), which represent an atypical B cell subset associated with various autoimmune diseases, showed a significant decrease in frequencies and a correlation with the MMT8 score. Within the DN subset, the DN1 subpopulation (CD21+ CD11c−) showed a significant decrease compared to the control group. There was also a significant decrease in the memory B cell subpopulation (CD27+ CD38−) and the non-classic memory B cell subpopulation (CD27− CD38− CD24+). A significant increase in naive B cells (CD27− IgD+) was observed. However, none of the previously described subpopulations correlated with clinical features.

Conclusion

This study demonstrated a decrease in frequencies in the double negative B cell subset in adult IIM patients and showed a correlation with clinical features, which suggests that these B cells could be related to disease activity. UNAM-DGAPA-PAPIIT IN212122
Type 1 interferons promote anti-RBC antibody responses in interferon-dependent lupus mice
Authors: Paul Kausik , Lee June Young , Hernandez-Armengol Rosario , Shibata Tomohiro , Jefferies Caroline Anne , Gibb David R
Published: August 2023

Red blood cell (RBC) transfusion can induce anti-RBC antibodies that promote hemolytic events. Patients with systemic lupus erythematosus (SLE) have an increased frequency of RBC alloimmunization, and two-thirds of patients express a type 1 interferon (IFNα/β) gene signature. We previously reported that IFNα/β induces RBC alloimmunization in the IFNα/β-dependent pristane lupus mouse model. However, it is unclear whether the lupus-like phenotype or IFNα/β directly promoted alloimmunization. Thus, we examined alloantibody responses in MRL/lpr mice, which have an IFNα/β-independent lupus-like phenotype. MRL/lpr mice produced anti-dsDNA lupus antibodies. However, unlike pristane-treated WT mice, IFNα/β-stimulated genes (ISGs) were not elevated in MRL/lpr mice. After transfusion with allogeneic RBCs, untreated WT and MRL/lpr mice produced minimal levels of anti-RBC antibodies, compared to elevated levels produced by pristane-treated WT mice. To determine whether IFNα/β can induce alloimmunization in MRL/lpr mice, WT and MRL/lpr mice were treated with recombinant IFNα (rIFNα) or the IFNα/β stimulus, poly(I:C), prior to transfusion. While rIFNa and poly(I:C) induced RBC alloimmunization in WT mice, they failed to induce alloimmunization in MRL/lpr mice. Thus, MRL/lpr mice are resistant to IFNα/β-induced alloimmunization due to a currently explored mechanism. These findings also illustrate that allogeneic transfusion induces alloimmunization in an IFNα/β-dependent lupus model, but not in an IFNα/β-independent model. Thus, the lupus-like phenotype is not sufficient to induce alloimmunization. Collectively, these findings warrant investigation of the role of IFNα/β in RBC alloimmunization in patients with SLE.

NIH (R01 HL165169-01); NIH (R03 HL158637-01); NIH (K08 HL141446-03)
TLR7 expression increases with disease progression and mediates expansion of distinct B cell subsets in primary Sjögren’s Disease
Authors: Punnanitinont Achamaporn , Kasperek Eileen M , Kramer Jill M
Published: August 2023

Primary Sjögren’s Disease (pSD) is a systemic autoimmune disorder in which the exocrine tissue exhibits chronic inflammation. Additionally, patients often experience diminished tear and saliva production along with many extra-glandular disease manifestations. The causes of pSD are poorly understood and no curative treatments are available. Previous studies in our lab conducted in a pSD mouse model demonstrated that TLR7 agonism accelerates pSD pathogenesis and drives expansion of age-associated B cells (ABCs). To extend our prior work, we hypothesized that pSD splenocytes show heightened TLR7 expression and activation, and expansion of B cell subsets linked to autoimmunity relies on TLR7 in pSD mice. We harvested the spleens of pSD females at three different disease time points (pre-disease, clinical disease and advanced stage disease) and performed flow cytometry. Parallel experiments were performed using age and sex-matched healthy controls. Splenocytes were also cultured with the TLR7 agonist Imiquimod (Imq) and IL-6 secretion assessed by ELISA. In addition, spleens were isolated from TLR7-deficient pSD females at the clinical disease stage and flow cytometry was performed. We found splenocytes derived from pSD animals at the clinical disease stage secreted higher levels of IL-6 in response to Imq stimulation as compared to those derived from healthy control animals. TLR7+ B cells and ABCs (B220+, CD23−, CD21−, T-bet+) increased with disease progression in pSD mice. In pSD mice lacking TLR7, ABCs and germinal center B cells were decreased compared to the TLR7-sufficient pSD parental strain. Thus, splenocytes from pSD mice show enhanced sensitivity to TLR7 and TLR7 signaling mediates expansion of ABC subset in pSD.

NIH/NIDCR T32 DE023056 NIH/NIDCR R01 DE029472
B cells Regulation by Interferon-gamma in a Neuromyelitis Optica Spectrum Disorder animal model
Authors: Arellano Gabriel , Loda Eileah , Chen Yanan , Podojil Joseph R , Neef Tobias , Popko Brian , Balabanov Roumen , Miller Stephen D
Published: August 2023

Neuromyelitis Optica Spectrum Disorder (NMOSD) is an autoimmune disease of the central nervous system (CNS) mediated by Th17 and auto-antibody response against the water channel protein Aquaporin-4 (AQP4). In NMOSD, B cells plays a central role regulating autoreactive T and antibody secreting cells, and the use of anti-B cells therapy is a common practice for patients. Despite this, the mechanisms of B cells response against AQP4 are poorly understood. We have demonstrated that the immune tolerance against the immunodominant AQP4201–220 peptide in mice is maintained by IFN-g, controlling disease incidence, severity, and chronicity. Actively immunization of C57BL/6 (WT) treated with neutralizing anti-IFN-g antibodies, IFN-g knockout (IFNGKO) and IFN-g receptor KO (IFNRKO) mice was induced using the AQP4201–220 peptide, showing an ascendant paralysis starting from the tail to complete hind limb paralysis. CNS histological analysis showed an increased presence of T and B cells, and the complement system in lesion sites, associated with CNS damage. CNS Th17 profile was also observed in the anti-IFN-g treated group. Conversely, IFN-g treatment in IFNGKO mice decrease disease severity with a reduction of CNS CD4+ and B cells. Remarkably, IFN-g regulates the IL-6 expression by B cells in an antigen specific fashion. B-cell depletion significantly reduced the disease incidence, T cells CNS infiltration and anti-AQP4201–220 serum antibodies. Finally, using anti-IL-6R treatment, we showed that IL-6 signaling is necessary for the B cell activation and their induction of Th17 cells. In conclusion, AQP4201–220 tolerance is strictly regulated by IFN-g and its signaling, restraining B cells pathogenicity through IL-6 signaling modulation.

This work was supported by National Institutes of Health grants R21AI151438 and R01 NS099334
Treg cell depletion in adult mice results in activation of antigen-presenting cells prior to fatal autoimmune disease
Authors: Kim Yong-Hee , Panda Abir K , Shevach Ethan M
Published: August 2023

Studies in mice expressing the diphtheria toxin receptor (DTR) exclusively on regulatory T (Treg) cells (Foxp3-DTR mice) demonstrated the critical importance of Treg cells in maintaining normal immune homeostasis. Adult Foxp3-DTR mice injected with DT begin to die from day 10 after DT treatment. Marked expansion of almost all immune cell types was observed prior to death. The purpose of the present studies was to examine in-depth changes in lymphocytes and antigen-presenting cells at early time points after Treg depletion to determine the most critical cell types controlled by Treg in the steady state. Complete depletion of Treg was observed 2 days after treatment, but profound activation of CD4+ and CD8+ T cells as measured by induction of CD44 expression and vigorous proliferation as measured by Ki-67 incorporation were not seen on day 2 but seen on day 6 of DT treatment. Curiously, most of the CD44hi Ki-67+ T cells were CD25− and plasma levels and intracellular levels of IL-2 were not increased suggesting that early activation of CD4+ and CD8+ T cells was not the result of a burst of IL-2 production. Increased expression of CD80, CD86, CD40, and PD-L1 could be detected on CD11c+ dendritic cells (DCs) on day 6. Anti-CD80/CD86 mAbs reversed the activation of T cells provoked by depletion of Treg cells, while anti-IL-2 or anti-IL-2Rb mAbs had no effect. Taken together, these studies suggest that disruption of the regulation of Treg-mediated control of DC activation represents the first step in the complex autoimmune disease seen after Treg depletion.

This work was supported by the Intramural Research Program of NIAID, NIH.

This work was supported by the Intramural Research Program of NIAID, NIH.
Chaperone-mediated autophagy modulates regulatory T cell differentiation and function
Authors: Harrison Ranee , Botbol Yair , Sidoli Simone , Cuervo Ana-Maria , Macian Fernando
Published: August 2023

Autophagy has been shown to regulate many key functions of the immune system including important roles in effector and memory T cell maintenance and function. Our group showed that chaperone-mediated autophagy (CMA), a selective type of autophagy that declines with age, plays a key role in T cell activation. In this work, we have analyzed the role of CMA in generation and function of regulatory T cells (Tregs). We found that resting Tregs display high basal CMA activity, which further increases upon Treg activation. We generated Treg specific CMA-deficient mice (Foxp3:Cre-LAMP2Afl/fl) and found that they have significantly reduced body weight due to reduced fat mass. We propose that the CMA status of adipose tissue infiltrating Tregs may have a direct impact on adipose tissue inflammation state. In fact, as the Treg-specific CMA-deficient mice age, we found marked inflammation and reduced survival, suggesting a role for CMA in maintaining Treg self-tolerance under homeostatic conditions. We confirmed that CMA-deficient Tregs have reduced suppressive activity in vivo and have identified the Treg sub-proteome regulated by CMA using comparative quantitative proteomic analysis of control and CMA-deficient Tregs. This has allowed us to identify cellular pathways behind the mechanism by which CMA regulates Treg function. The proteomic analysis hits will help in identifying potential targets of CMA that could be used therapeutically to modulate Treg function, to prevent autoimmunity or boost anti-tumor immune responses.

Supported by grants from NIH

P01-AG031782

R01-AI113919
Social Status is Associated with Impaired Anti-Inflammatory Response in Free-Ranging Male Rhesus Macaques
Authors: Sanchez Rosado Mitchell R , Newman Laura , Watowich Marina , Pavez-Fox Melissa , Valle Josue Negron-Del , Phillips Daniel , Skelton Macaela , Siracusa Erin , Higham James , Brent Lauren , Lea Amanda , Sariol Carlos , Snyder-Mackler Noah
Published: August 2023

Exposure to social adversity can influence the onset and progression of disease. One possible mechanism through which social adversity may impact health outcomes is by altering immune homeostasis. Recently, evidence has connected social adversity to changes in gene regulation in the immune response of both humans and nonhuman primates. We investigated how social adversity (quantified as low social status) affected the Th2 immune response in free-ranging male rhesus macaques (n=128). We stimulated peripheral blood mononuclear cells (PBMCs) in vitro with Dexamethasone (Dex), an anti-inflammatory synthetic glucocorticoid (GC), and measured how social status influenced the gene regulatory response to Dex stimulation using mRNA-seq. We detected 2,923 differentially expressed genes (FDR<0.1) in response to Dex in PBMCs. After Dex stimulation, glucocorticoid responsive genes, such as FKBP5 and TSCD22, were induced and inflammatory genes, such as CSF1 and TNF, were repressed. Low social status animals had significantly higher mean expression of inflammatory genes (p=1.2×10−20) compared to high social status animals. Many of these genes, such as CD38 and NKAP, are known to be repressed by Dex. After Dex stimulation, low social status animals also had a higher mean expression (p=1.9×10−4) of genes related to the GC resistance-associated c-Myc/MAPK pathway. For instance, low social status animals had significantly higher expression (FDR<0.2) of signature genes in this pathway including AKT3, MAPK1 and PIK3CD. Our study suggests that the anti-inflammatory immune response in rhesus macaques can be negatively affected by low social status, with animals having a blunted response to Dex and presenting signatures of GC resistance.

R01-AG060931 R00-AG051764 P40-OD012217
Host-targeted self-attenuated influenza virus as an effective nasal spray vaccine in aged population
Authors: De La O Perez Rodrigo , Chen Yanping , Zeng Mingtao
Published: August 2023

The elderly are at high risk for influenza infection and its complication. However, current inactivated influenza vaccines are often modestly immunogenic and show low protective immunity in the elderly, due to immunosenescence. New approaches are urgently needed for developing effective vaccines against influenza for the elderly. In current study, we have constructed new recombinant influenza viruses, named host-targeted self-attenuated influenza viruses (SAIVs), which can express functional mammalian species-specific artificial microRNAs (amiRNAs). The expression of these amiRNAs can inhibit expression of some host factors critical for influenza replication, and therefore the resultant recombinant influenza viruses are replication restricted and attenuated in the host cells. One of these SAIVs, which can express an amiRNA that inhibits expression of the host cellular Cdc2-like kinase 1 (CLK1), was produced in embryonic chicken eggs and evaluated in an aged mouse model of influenza infection. It elicited robust antibody and T cell responses against influenza virus and demonstrated significantly protective efficacy against lethal infection with wild type influenza virus H1N1 PR8 after single dose of intranasal vaccination. Our research finding provided a proof of concept that the new host targeted self-attenuated influenza virus can be further used as an effective vaccine against influenza in aged population.

Supported by a research grant from National Institute of Allergy and Infectious Diseases (AI133207).
Engineered cytokines target atherosclerotic plaques and locally suppress inflammation
Authors: Volpatti Lisa R , Reda Joseph , Borjas Gustavo , Zhou Zhengjie , Fang Yun , Hubbell Jeffrey
Published: August 2023

Chronic inflammatory diseases are often treated with corticosteroids or TNF blockers to suppress overactive immune responses. However, these immunosuppressants also dampen healthy immune responses to opportunistic pathogens (e.g., respiratory viruses) and are associated with adverse effects. Targeted immunotherapies are needed to provide local immunosuppression without systemic effects. In the context of atherosclerosis, the anti-inflammatory cytokine IL-10 has been shown to suppress vascular inflammation, but its poor pharmacokinetic profile and pleiotropic effects have limited its therapeutic potential. To overcome these challenges, we engineered IL-10 to specifically target atherosclerotic plaques. We accomplished this goal by constructing fusion proteins in which one side is IL-10 and the other side is an antibody fragment (Fab) that binds to protein epitopes of low-density lipoprotein (LDL). In murine models of atherosclerosis, we show that systemically administered Fab-IL-10 constructs bind circulating LDL and hitchhike a ride to atherosclerotic plaques. In a biodistribution study, we observe elevated levels of IL-10 in the aorta but not the lungs of hyperlipidemic mice, indicative of targeted delivery. The targeted Fab-IL-10 constructs significantly reduce aortic immune cell infiltration to levels comparable to healthy mice, whereas non-targeted IL-10 has no therapeutic effect. Mechanistically, we demonstrate that Fab-IL-10 constructs are preferentially taken up by macrophages to exert an anti-inflammatory effect. This platform technology can be applied to a variety of cytokines and shows promise as a potential targeted anti-inflammatory therapy.

Supported by the Chicago Immunoengineering Innovation Center, the NIH National Heart, Lung, and Blood Institute, and the American Heart Association.
Defining the features facilitating accurate prediction of antibodies binding to the same epitope
Authors: Jarjapu Mahita , Peters Bjoern
Published: August 2023

Antigen specificity of antibodies is defined mainly by their complementarity-determining regions (CDRs) which show high variability in their sequences and structures. Antibodies having identical CDR sequences typically bind to the same epitope on an antigen. The conventional paradigm is that the function, including the epitope specificity, of an antibody is determined by its structure. However, we wanted to estimate how much additional information is contained in an antibody’s structure, relative to its sequence alone. High-throughput sequencing (HTS) technologies and advances in structural determination methods have led to an increased amount of antibody sequence and structural data in recent years. Here, we set out to leverage the large body of publicly-available antibody data to identify features that are most informative in predicting if two antibodies are likely to bind to the same epitope. We utilized three datasets of antibody pairs to develop machine learning (ML) models for investigating the contributions of sequence and structural features in predicting if two antibodies are likely to target the same epitope. Sequence features included pairwise sequence identities, while structural features included CDR structural similarity as well as the geometrical similarity of antibody paratopes. Surprisingly, features based on CDR sequence identity alone seemed to be sufficient to predict whether a pair of antibodies bind to the same epitope. Inclusion of CDR or paratope structural information in addition to CDR sequence information did not lead to major improvements in the predictive performance of the ML models. These findings suggest that antibody CDR sequences retain most of the information about antigen specificity.

National Institute of Allergy and Infectious Diseases of the National Institutes of Health
Immunogram: a Generalized Framework to Measure a Person’s Immune System
Authors: Medetgul-Ernar Kwat , Mohsin Azam , Davis Mark M
Published: August 2023

“How is my immune system?” This is a simple question, but there is no metric to assess the overall performance of the human immune system. Current clinical methods that evaluate a person’s immune system only provide limited information, like cell counts or immunoglobulin levels, and fail to capture the global immune state. This project aims to develop Immunogram, a generalized framework to measure a person’s immune system. Immunogram is a Julia-based software that links cells, proteins, and other molecules in a knowledge graph. Immunogram delineates subgraph patterns that describe human immune states and uses them to characterize a person’s immune system. Immunogram can be used to identify immune cell types, predict vaccine response, and determine if a person has an ongoing infection.

Kwat Medetgul-Ernar was supported by Stanford University Medical Scientist Training Program grant T32-GM007365 and T32-GM145402.
Modeling the SARS-CoV-2 receptor ACE2 indicates that protein-bound Zn affects distant protein stability and interactions
Authors: Fatouros Peter R , Roy Urmi , Sur Shantanu
Published: August 2023

Molecular Dynamics (MD) is a widely used drug design tool capable of rapidly screening the binding capacity of many candidate inhibitors in a cost-effective manner. This approach has been leveraged to search for molecules that can bind to angiotensin converting enzyme 2 (ACE2) and prevent SARS-CoV-2 viral entry to host cells. However, ACE2 is difficult to model due to an embedded zinc ion (Zn), which introduces complex charge transfer and polarization. Since the embedded Zn is distant from the site of ACE2 binding, many groups have assumed Zn does not impact protein interactions and have excluded Zn while modeling ACE2. Here, we evaluated the impact of Zn by performing MD simulations of Zn-bound and Zn-free versions of the ACE2-spike protein (S1) and ACE2-monoclonal antibody (mAb) systems . We found that excluding Zn had a significant effect on total protein stability and the stability of interacting residues, indicating that Zn can impact distant protein sites. Additionally, we discovered that including protein-bound Zn in MD simulations improved the binding free energy of both systems by −3.26 and −14.8 kcal/mol in the ACE2-S1 and ACE2-mAb systems, respectively. These data suggest that excluding Zn may alter inhibitor selection decisions and should be included for accurate modeling. Interestingly, ACE2-mAb is particularly sensitive to changes in receptor structure, likely reflecting its generation against in vivo Zn-bound ACE2. Collectively, our data demonstrates that excluding Zn significantly impacts MD simulation outcomes which inform inhibitor selection, particularly when modeling receptor-mAb complexes.
Comparison of CAR-T cell-mediated cytotoxicity assays with suspension tumor cells using high-throughput plate-based image cytometry method
Authors: Chan Leo , Sun Yu-Jun , Chen Yi-Chun , Hua Wei-Kai , Wu Sariena
Published: August 2023

In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. Currently, there are six CAR-T cell products available in the market including Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykt. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Traditionally, in vitro CAR-T cell-mediated cytotoxicity is assessed using release assays such as 51Cr (radioactivity), calcein (fluorescence), and LDH (enzymatic). Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo high-throughput plate-based Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. Performing time- and E:T ratio-dependent CAR-T cell-mediated cytotoxicity assays, the GFP-based method demonstrated higher sensitivity in detecting the level of cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions.

None
A novel approach for identification of highly potent pathogen specific human CD8 T cells from peripheral blood
Authors: Anikeeva Nadia , Sykulev Yuri
Published: August 2023

Measuring frequency and quality of antigen-specific T cells serves as essential indicators for diagnostic and prognostic outcomes of the immune response. We have developed a novel assay allowing to measure the frequency and quality of individual antigen-specific T cells within polyclonal population of CD8 T cells assembled into tight monolayer. The assay determines kinetics and pattern of Ca2+ mobilization in every individual cell. Those T cells demonstrating rapid and sustained Ca2+ influx are highly potent and capable to rapidly destroy target cells. This has been confirmed by testing responses of HIV-specific CD8 T cells against variants of HIV-derived peptide ILKEPVHGV that induce responses of different potency. These data were consistent with comparison of HCMV-specific CD8 T cells from healthy donors and people demonstrating HCMV reactivation followed bone marrow transplantation. Vast majority of the CD8 T cell from healthy adults revealed rapid and sustained Ca2+ response. HCMV-reactivation was associated with a large percentage of CD8 T cells demonstrating oscillating Ca2+ mobilization while fraction of the T cells showing rapid Ca2+ response was very small. Our goal is to isolate potent T cells and sequence their genes encoding TCR that could be utilized for engineering of highly potent T cells to attack pathogens and cancer cells.

Supported by grants from NIH (U01 AI148117)
T cell functional phenotyping on a single cell scale using hydrogel microparticles
Authors: Kizerwetter Monika , Koo Doyeon , Di Carlo Dino , Spangler Jamie B
Published: August 2023

Engineered cell therapies, especially chimeric antigen receptor (CAR) T cell therapies, have shown much promise in treating various cancers, most notably hematologic malignancies. However, the success of these therapies is highly variable between cancer types and across patient populations. A growing body of literature suggests that certain T cell functional properties are key drivers of response; thus, it is imperative to develop a more thorough understanding of the functional heterogeneity in T cell populations in order to inform the design of the next generation of cancer immunotherapies. Here, we used a new technology our team developed known as nanovials, which are microfluidically manufactured hydrogel microparticles, to enable the specific activation and capture of secreted products of T cells on a single-cell level. Leveraging this platform, we characterized the secretion of IFN-γ and TNF-α by individual CD8+ T cells harvested from OT-I mice. After initial expansion after isolation, T cells were loaded into functionalized nanovials and specifically activated with OVA peptide. The cells loaded in the nanovials were then separated based on cytokine secretion levels via fluorescence-activated cell sorting (FACS). Unlike traditional cytokine secretion assays which require fixation and permeabilization, cells remain viable throughout our process, allowing for sorted subpopulations to be dissociated from the microparticles, expanded, and further analyzed via cytotoxicity, co-culture, or phenotypic studies. Collectively, our efforts introduce a new paradigm for elucidating the functional properties of immune cells, providing critical insights that will guide the future design of engineered cell therapeutics.

M.K. is a recipient of a National Science Foundation Graduate Research Fellowship Program award.
Characterization of germinal center and circulating T follicular helper cells using both intracellular and surface CITE-seq
Authors: Cashman Siobhán B , Swift Teresa , Song Hye-Won , Wright Adam , Ayer Aruna
Published: August 2023

T follicular helper (TFH) cells are a CD4+ T cell subset that play a critical role in the formation of germinal centers (GC). TFH cells are essential to produce protective antibody responses through regulation of clonal selection & differentiation into activated antibody-secreting & memory B cells. TFH cell differentiation is a multistep process that begins with presentation of antigen by dendritic cells to naïve CD4+ T cells, where CD4+ T cells commit to the TFH lineage by expressing the lineage defining transcription factor B cell lymphoma 6 (Bcl-6). TFH cells are phenotypically characterized by high expression of C-X-C chemokine receptor 5 (CXCR5) & programmed cell death-1 (PD-1) on the cell surface. CXCR5+ memory CD4+ T cells present in peripheral blood represent a circulating TFH (cTFH) cell subset. In contrast to TFH cells, cTFH cells may express low levels of Bcl-6 with numerous reports of subsets that differ both phenotypically & functionally. Here we characterise the heterogeneous subsets of TFH & cTFH cells using a combination of intracellular CITE-seq, surface CITE-seq and whole transcriptome single-cell RNA sequencing (scRNA-seq) analysis. Germinal center and circulating TFH cells were enriched by sorting memory CD4+, CXCR5+, PD-1+, and memory CD4+ CXCR5+ cells, respectively. We examined the expression of Bcl-6 & other transcription factors, using protein and RNA expression data, across pre-TFH, TFH & cTFH cells. Together, these data demonstrate how a combination of intracellular CITE-seq, surface CITE-seq & scRNA-seq may be used to deepen our understanding of the differentiation states & subsets of cells present at low abundance, by enabling detection of intracellular and surface protein along with high-quality mRNA.
Design and Validation of Ultrahigh-plex Discovery Panels for Immune Checkpoint Targets
Authors: Duerrschmid Clemens , Mukherjee Avik , He Yan , Berry Shannon , Shang Olive , McLane Michael , Ihley Nicholas , Zheng Yi
Published: August 2023

Ultrahigh-plex single-cell spatial phenotyping is revolutionizing immuno-oncology research. To enable in-depth characterization of the tumor microenvironment (TME), we have developed and validated pre-optimized antibody panels on cancer tissues to reveal key cell types and cellular architecture using the PhenoCycler™-Fusion system.

We designed multiple panels that answer specific biological questions about the TME, and once multiplexed together create a comprehensive view of the structure and function of the TME. The resulting PhenoCode™ Discovery panels include markers that identify immune cell types, activation states, immune checkpoints, and mediators of proliferation. Each antibody in the panel was conjugated to an oligo barcode and stained on human FFPE tissue. Staining specificity of each conjugated antibody was assessed by comparing immunofluorescent results from PhenoCycler™ with a DAB chromogenic immunohistochemistry assay. The slide stained with a PhenoCode™ Discovery panel was imaged on a PhenoCycler™-Fusion platform, where dye-labeled oligo reporters (complementary to the barcodes) are hybridized to the barcode to visualize the antibody. Antibody concentration, exposure time, and corresponding dye were further optimized, verified, and validated as a whole module using tonsil and cancer tissues based on image analysis and intensity analysis.

In this study, we showcased the rigorous process used for development and validation of PhenoCode™ Discovery panels and demonstrated how they can work together to collect comprehensive data from one tissue sample. The introduction of these pre-optimized antibody panels marks a pivotal point in the evolution of ultrahigh-plex spatial phenotyping.
Multi-pass flow cytometry using laser particle barcodes for easy high-marker analysis
Authors: Kwok Sheldon J , Hyun William , Forward Sarah , Fahlberg Marissa , Abbott Geoffrey , Assita Emane Rose , Zhu Tony
Published: August 2023

Current flow cytometry requires samples to be fully processed and stained for all markers of interest before acquisition. However, assay development become increasingly difficult when many colors (>10) are used at once due to spectral spillover. Assay complexity is hampered further by markers that require special cell processing and restrict the choice of fluorophores or antibodies.

We have developed a method to dramatically simplify high-marker flow assays by breaking down a complex measurement into multiple, easier measurements. Our method involves optically barcoding individual cells with semiconductor-based laser particles (LPs) that emit laser light in the infrared. Using LP barcodes and a custom flow cytometer, we track millions of cells across repeated measurements or passes. Cells are de-stained and re-stained after each pass.

We demonstrate several applications of multi-pass flow cytometry. First, we show deep immunophenotyping of human PBMCs, measuring 33 markers with 13 colors over 3 cycles. We validate our method against state-of-the-art, reproducing expected cell types and showing improved data quality due to significantly reduced spillover. Second, we show deep characterization of stimulated mouse T cells, in which an unprecedented 32 markers including activation markers, intranuclear proteins, and intracellular cytokines are detected on the same cell type. Finally, we measure cells before and after harsh cell processing steps to recover signals that would otherwise be lost, such as concurrent detection of cytoplasmic GFP and cell cycle.

Multi-pass flow cytometry reduces the barrier to high-marker analysis by simplifying complex panels and enabling detection of sensitive signals without compromise.

Supported by NIH grant R44GM139504
StarBright Dyes:-Making Panel Design Easier For Everyone
Authors: Blundell Michael , Sanderson Sharon
Published: August 2023

Following simple rules during multicolor panel building can help alleviate some common problems when immunophenotyping common immunological samples. However, building large panels can still be challenging.

Bio-Rad’s new StarBright Dyes have been specifically designed for use in multicolour conventional and spectral flow cytometry to help alleviate common challenges such as brightness, spillover, fixation and buffer compatibility. Data we show here reveals how StarBright Dyes can help make panel design easier for everyone.
Longitudinal somatic mutation analysis of individual human CD8+ T cells by a UMI-based single cell mutation detection (USCMD) method
Authors: Hemani Humza , Ahmad Raheel , Cifello Jeffrey , Li Siyi , Li Jiangyuan , Lu Jian , Ellabaan Mostafa , Weng Nanping
Published: August 2023

Somatic mutation is a contributing factor for cancer and aging. DNA-based single-cell mutation detection methods have been developed, but are hindered by the limited number of cells examined as well as by errors from PCR amplification and sequencing methods. Here, we developed a UMI-based scRNAseq mutation detection (USCMD) method that allows for the examination of up to 10,000 cells per sample with reduced PCR and sequencing errors, and which also filters potential errors of RNA polymerase II. We applied USCMD to analyze the scRNAseq data from human CD8+ T cells collected from 8 donors with 2 visits over a period of 9 years (aged from 30 to 69 years at first visit). We found that some novel missense mutations were associated with changes (increase or decrease) in the number of cells carrying them over time, and were able to identify clonally related cells based on shared somatic mutations. Together, this USCMD method improves the accuracy of mutational analysis of scRNAseq data and allows parallel transcriptional assessment of mutated and unmutated cells to understand their potential functional consequences.