Tag: Antigen

Xenotransplantation model used for studying microglial response to Alzheimer’s disease

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Microglia are specialized immune cells in the brain. While they normally protect our brains, they can also contribute to neurodegenerative diseases such as Alzheimer’s. The exact mechanism behind this contribution is not yet fully understood due to the complexities involved in studying them in human brain samples. Now, a research team led by Prof. Bart De Strooper (UK-DRI@UCL and VIB-KU Leuven) and Prof. Renzo Mancuso (VIB-UAntwerp) made a xenotransplantation model – mice with stem-cell-derived human microglia in their brains to observe how human microglia respond to the disease environment. Their findings, published in Nature Neuroscience, will help scientists better understand the complex mechanisms involved in Alzheimer’s disease.

Alzheimer’s disease (AD) is a complex, progressive neurodegenerative disorder that affects millions of people worldwide. The World Health Organization predicts a tripling of cases by 2050, highlighting the urgent need for new treatments.

Microglia, our brain’s immune cells, are responsible for clearing debris and responding to inflammation in the brain. Scientists have been studying these cells in AD, as they play a central role in the disease, especially in the building-up of and early response to amyloid-β plaques, a hallmark of the disease. The microglia react to the plaques as they are perceived as foreign to the brain, making them the main drivers of the neuroinflammation that characterizes AD. Studying these cells in human brain samples post-mortem can be challenging because of genetic differences between people, the time between death and examination, and the presence of other brain disorders. Indeed, studies in human postmortem brain samples have shown mixed results regarding the reaction of microglia. It’s also not possible to test the effects of medication on post-mortem brains.

That is why the first authors of the study, Dr. Nicola Fattorelli and Dr. Anna Martinez Muriana, together with their colleagues at the VIB-KU Leuven Center for Brain & Disease Research, the VIB-UAntwerp Center for Molecular Neurology and the UK Dementia Research Institute, developed a unique mouse model. This xenotransplantation model is genetically engineered to mimic the amyloid-β plaque accumulations seen in humans with AD and can be transplanted stem-cell-derived human microglia. Previously, a similar model was able to show how transplanted human neurons die in AD. Now, this approach allowed the researchers to investigate how human microglia respond to amyloid plaques during the course of the disease.

Microglial responses to AD

The scientists found that human microglia showed a much more complex immune response to amyloid-β than their rodent counterparts. Human microglia also displayed a different genetic transition from the normal to the reactive state of the cells.

This could have implications for the development of treatments. Researchers need to be cautious when using mouse models to study AD in preclinical systems for potential therapeutic targeting of microglia because the responses of human and mouse microglia may not be the same.”

Professor Renzo Mancuso, first author of the study and group leader at the VIB-UAntwerp Center for Molecular Neurology

Genetics and early intervention

The study also revealed that different genetic risk factors for AD influence how human microglia respond to the disease. Moreover, the genetic risk of AD was spread over the different reactive states of the microglia, further demonstrating the importance of microglia in the disease process. This suggests that future microglia-targeted therapies need to be implemented with care as genetic factors might differentially affect their cell states and modify the disease course in unpredictable ways.

Furthermore, the data hinted at a possible interaction between microglia and soluble forms of amyloid-β, which appear early in the disease process, well before plaques form. This interaction might occur in the very early stages of Alzheimer’s and could potentially influence how the disease progresses. The question remains as to whether this microglial response affects neurons or other brain cells, inducing the cellular responses in AD that ultimately result in neurodegeneration, and what this means for possible treatments. In the meantime, this model provides a unique possibility to test novel drugs against human microglia for the treatment of AD.

“Overall, this research is an important step toward understanding the mechanisms behind AD. The study provides new insights into the complex ways human microglia respond to AD, which could help researchers develop better treatments for the disease,” concludes Professor De Strooper. “Our findings validate this xenograft model as a powerful tool to investigate the genetics underlying microglial response in Alzheimer’s.”

Source:

Vlaams Instituut voor Biotechnologie

Journal reference:

Mancuso, R., et al. (2024). Xenografted human microglia display diverse transcriptomic states in response to Alzheimer’s disease-related amyloid-β pathology. Nature Neuroscience. doi.org/10.1038/s41593-024-01600-y.

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March 27, 2024
Scientists aim to understand why T cells do not sustain energy in tumors

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T cells are often called “assassins” or “killers” because they can orchestrate and carry out missions to hunt down bacteria, viruses, and cancer cells throughout the body. Mighty as they may be, recent research has shown that once T cells infiltrate the environment of a solid tumor, they lose the energy needed to combat the cancer.

A research team led by Jessica Thaxton, PhD, MsCR, associate professor of cell biology and physiology and co-leader of the Cancer Cell Biology Program at the UNC Lineberger Comprehensive Cancer Center, aimed to understand why T cells do not sustain energy in tumors. Using their expertise in tumor immunity and metabolism, the Thaxton Lab, led by the Katie Hurst, MPH, and 4^th year graduate student Ellie Hunt, found that a metabolic enzyme called Acetyl-CoA Carboxylase (ACC) causes T cells to store fat rather than burning fat for energy.

Our discovery fills a long-standing gap in knowledge regarding why T cells in solid tumors don’t appropriately generate energy. We inhibited the expression of ACC in mouse cancer models, and we observed that T cells were able to persist much better in solid tumors.”

Jessica Thaxton, PhD, MsCR, associate professor of cell biology and physiology and co-leader of the Cancer Cell Biology Program at the UNC Lineberger Comprehensive Cancer Center

The new findings and immunotherapeutic strategies, which were published in Cell Metabolism, could be used to make multiple types of T-cell therapies more effective for patients, possibly encompassing both checkpoint and chimeric antigen receptor (CAR) T-cell therapies.

In the field of cancer immunotherapy, it has long been known that T cells are not able to create their cellular energy, called adenosine triphosphate or ATP, when they are inside of a solid tumor.

In 2019, Thaxton’s lab studied a T cell with optimal antitumor function. In a publication in Cancer Immunology Research, Hurst and Thaxton used a proteomics screen to identify enzymes associated with the optimal antitumor metabolism of these T cells. Through this screen, the two discovered that ACC expression may limit the ability of T cells to make ATP in tumors. ACC, a key molecule that is involved in many metabolic pathways, blocks cells from breaking down fat and using it as fuel for energy in mitochondria.

“Acetyl-CoA carboxylase can drive the balance between storing lipids versus breaking down those lipids and feeding them into the citric acid cycle for energy,” said Thaxton. “If ACC is flipped ‘on’, cells generally store lipid. If ACC is ‘off’, cells tend to use the lipid in their mitochondria to make ATP.”

Using Hunt’s expertise in confocal imaging, the research team was able to observe lipid stores in T cells isolated from multiple types of cancers. The observation, as well as other experiments, confirmed the team’s hypothesis that T cells were storing lipids instead of breaking them down.

Thaxton’s team then used CRISPR Cas9-mediated gene deletion to see what would happen if they “deleted” ACC from the picture. There was a rapid reduction in the amount of lipid storage in T cells, and the team was able to visualize fat relocating to the mitochondria to be used to generate energy.

Thaxton now hypothesizes that T cells may need a “delicate balance” of lipids to persist in solid tumors with a certain amount of lipid dedicated to cancer cell assassination and low levels of fats being maintained in stores.

The latest findings could prove to be useful in enhancing chimeric antigen receptor (CAR) T-cell therapies. This cutting-edge technology takes T cells out of cancer patients, modifies them in the lab to hunt down tumor cells, and then re-infuses the cells to fight the patient’s cancer. Preliminary data from Thaxton’s lab demonstrates that even the manufactured T cells contain excess lipid stores.

The lab is starting to look in patient samples to understand how researchers can possibly flip the ACC metabolic switch directly in patient tumors, negating the need to take out and reinfuse cells back into the body. But researchers must first determine how this could affect other immune cell populations in the body, such as macrophages.

Source:

University of North Carolina Health Care

Journal reference:

Hunt, E. G., et al. (2024). Acetyl-CoA carboxylase obstructs CD8+ T cell lipid utilization in the tumor microenvironment. Cell Metabolism. doi.org/10.1016/j.cmet.2024.02.009

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March 15, 2024
CHOP study uncovers metabolic key to faster immune response

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Researchers from Children’s Hospital of Philadelphia (CHOP) identified a key metabolite in cells that helps direct immune responses and explains at a single cell level why immune cells that most efficiently recognize pathogens, vaccines, or diseased cells grow and divide faster than other cells. The findings also indicate that a better understanding of this metabolite and its role in immune response could improve the design of immunotherapies and create longer-lived responses against different types of cancer as well as enhance vaccine strategies. The findings were published online today by the journal Science Immunology.

Antigens are foreign substances that our immune system recognizes and responds to by producing more T and B cells. These cells each have unique receptors that recognize specific antigens and can respond appropriately, and they can “remember” and respond similarly when exposed to the same antigen again. How well a T or B cell to sees its antigen is known as its affinity. This fundamental concept of immunology is how vaccines work. When those T and B cells encounter a pathogen, the body needs the ones that recognize their antigen the best, with high affinity, to divide more quickly to produce more daughter cells and “attack” the invader.

However, the underlying mechanisms as to why high affinity immune cells respond more efficiently have remained a mystery for researchers. After seeing an antigen, the chemistry inside T and B cells needs to change to allow them to properly respond. The researchers in this study wanted to look at metabolism to understand what causes high affinity cells to know that they need to divide more quickly to respond appropriately.

We wanted to see if specific metabolites were sensitive to T cell receptor affinity and controlled T cell expansion during immune responses.”

Will Bailis, PhD, Senior Study Author, Assistant Professor of Pathology and Laboratory Medicine at CHOP and the Perelman School of Medicine of the University of Pennsylvania

The researchers identified nicotinamide adenine dinucleotide (NAD) as a key, affinity-dependent component of T cell receptor metabolic reprogramming during the early stages of a T cell activation. Using flow cytometry, the researchers could look at NAD in single cells immediately after activation and show how it dictates the number of times T cells can divide in the future. Therefore, researchers could essentially predict how T cells behave and how many times they divide based on how much NAD they started with.

Additionally, the researchers found that manipulating how much NAD a cell was allowed to make could control when that cell went from a resting state to wanting to divide, suggesting that the metabolite could be used to improve response in certain T cell-driven therapies or vaccines.

“We believe this work shows how single cell differences in metabolism are a key reason why similar cells sometimes display strikingly different behaviors and that this may provide insight into underlying processes that drive disease and dysfunction that cannot simply be explained by gene regulation or signaling,” Bailis said. “With more work, we also believe that this information could potentially be used to improve vaccine strategies and the response and durability of cell-based therapies used to treat cancer and other diseases.”

This study was supported by National Institutes of Health grants K22AI141758, R35GM138085, R01DK098656, R01HL165792, P30ES013508, R01AI165706, and F31CA261156, a Children’s Hospital of Philadelphia Cell and Gene Therapy Collaborative SEED Award, a Children’s Hospital of Philadelphia Junior Faculty Pilot Grant, Transfusion Medicine Research Training Program grant 2T32HL00777528, Microbial Pathogenesis and Genomics Training Grant 5T32AI141393, and Immunobiology of Normal and Neoplastic Lymphocytes Training Grant T32CA009140.

Source:

Children’s Hospital of Philadelphia

Journal reference:

Turner, L., et al. (2024). Single-cell NAD(H) levels predict clonal lymphocyte expansion dynamics. Science Immunology. doi.org/10.1126/sciimmunol.adj7238.

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March 15, 2024
DNA origami vaccine DoriVac paves way for personalized cancer immunotherapy

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Therapeutic cancer vaccines are a form of immunotherapy in the making that could not only destroy cancer cells in patients, but keep a cancer from coming back and spreading. Multiple therapeutic cancer vaccines are being studied in clinical trials, but despite their promise, they are not routinely used yet by clinical oncologists to treat their patients.

The central ingredient of therapeutic cancer vaccines is antigens, which are preferentially produced or newly produced (neoantigens) by tumor cells and enable a patient’s immune system to search and destroy the cancerous cells. In most cases, those antigens cannot act alone and need the help of adjuvant molecules that trigger a general alarm signal in immune cells known as antigen-presenting cells (APCs). APCs internalize both antigen and adjuvant molecules and present the antigens to different types of T cells. Those T cells then launch an immediate attack against the tumor, or preserve a longer-lasting memory of the tumor for future defense.

A cancer vaccine’s effectiveness depends on the level and duration of the “alarm” its adjuvants can ring in APCs. Previously, researchers found that delivering adjuvant and antigen molecules to APCs simultaneously using nanostructures like DNA origami can increase APC activation. However, none of these approaches systematically investigated how the number and nanoscale arrangement of adjuvant molecules affect downstream tumor-directed immunity.

Now, a research team at the Wyss Institute at Harvard University, Dana-Farber Cancer Institute (DFCI), Harvard Medical School (HMS), and Korea Institute of Science and Technology (KIST) has created a DNA origami platform called DoriVac, whose core component is a self-assembling square block-shaped nanostructure. To one face of the square block, defined numbers of adjuvant molecules can be attached in highly tunable, nanoprecise patterns, while the opposite face can bind tumor antigens. The study found that molecules of an adjuvant known as CpG spaced exactly 3.5 nanometers apart from each other resulted in the most beneficial stimulation of APCs that induced a highly-desirable profile of T cells, including those that kill cancer cells (cytotoxic T cells), those that cause beneficial inflammation (Th-1 polarized T cells), and those that provide a long-term immune memory of the tumor (memory T cells). DoriVac vaccines enabled tumor-bearing mice to better control the growth of tumors and to survive significantly longer than control mice. Importantly, the effects of DoriVac also synergized with those of immune checkpoint inhibitors, which are a highly successful immunotherapy that is already widely used in the clinic. The findings are published in Nature Nanotechnology.

“DoriVac’s DNA origami vaccine technology merges different nanotechnological capabilities that we have developed over the years with an ever-deepening knowledge about cancer-suppressing immune processes,” said Wyss Core Faculty member William Shih, Ph.D., who led the Wyss Institute team together with first-author Yang (Claire) Zeng, M.D., Ph.D. “We envision that in the future, antigens identified in patients with different types of tumors could be quickly loaded onto prefabricated, adjuvant-containing DNA origami to enable highly effective personalized cancer vaccines that can be paired with FDA-approved checkpoint inhibitors in combination therapies.”

Shih is also a Professor at HMS and DFCI’s Department of Cancer Biology and, as some of the other authors, a member of the NIH-funded cross-institutional “Immuno-engineering to Improve Immunotherapy” (i3) Center based at the Wyss.

DNA origami rationale

The CpG adjuvant is a synthetic strand of DNA made up of repeated CpG nucleotide motifs that mimic the genetic material from immune cell-invading bacterial and viral pathogens. Like its natural counterparts, CpG adjuvants bind to a “danger receptor” called TLR9 in immune cells, which in turn induces an inflammatory (innate) immune response that works in concert with the antigen-induced (adaptive) immune response.

“We knew from previous work that to trigger strong inflammatory responses, TLR9 receptors need to dimerize and aggregate into multimeric complexes binding to multiple CpG molecules. The nanoscale distances between the CpG-binding domains in effective TLR9 assemblies revealed by structural analysis fell right into the range of what we hypothesized we could mirror with DNA origami structures presenting precisely spaced CpG molecules,” explained Zeng, who was an Instructor in Medicine at the time of the study and now is a senior scientist at DFCI and Harvard Medical School (HMS). In addition to Shih, Zeng was also mentored on the project by senior authors Ju Hee Ryu, Ph.D., a Principal Researcher at KIST, and Wyss Founding Core Faculty member David Mooney, Ph.D., who also is Professor at Harvard John A. Paulson School of Engineering and Applied Sciences (SEAS), and one of the i3 Center’s Principal Investigators.

Zeng and the team fabricated DoriVac vaccines in which different numbers of CpG strands were spaced at 2.5, 3.5, 5, or 7 nanometers apart from each other on one face of the square block, and a model antigen was attached to the opposite face. They protected their structures from being degraded in the body using a chemical modification method that Shih’s group had developed earlier. When internalized by different types of APCs, including dendritic cells (DCs), which orchestrate tumor-directed T cell responses, the DoriVac vaccines improved the uptake of antigens compared to controls consisting of free antigen molecules. A CpG spacing of 3.5 nanometers produced the strongest and most beneficial responses in APCs, and significantly outperformed a control vaccine containing only free CpG molecules. “We were excited to find that the DoriVac vaccine preferentially induced an immune activation state that supports anti-tumor immunity, which is what researchers generally want to see in a good vaccine,” said Zeng.

Besides spacing, the numbers of CpG molecules in DoriVac vaccines also mattered. The team tested vaccines containing between 12 to 63 optimally spaced CpG molecules and found that 18 CpG molecules provided the best APC activation. This meant that their approach can also help limit the dosage of CpG molecules and thus minimize commonly observed toxic side effects observed with adjuvants.

Gained in (tumor) translation

Importantly, these in vitro trends translated to in vivo mouse tumor models. When prophylactically injected under the skin of mice, DoriVac vaccines accumulated in the closest lymph nodes where they stimulated DCs. A vaccine loaded with a melanoma antigen prevented the growth of subsequently injected aggressive melanoma cells. While all control animals had succumbed to the cancer by day 42 of the experiment, DoriVac-protected animals all were alive. DoriVac vaccines also inhibited tumor growth in mice in which the formation of melanoma tumors was already underway, with a 3.5 nanometer spacing of 18 CpG molecules again providing maximum effects on DC and T cells, and the strongest reduction in tumor growth.

Next, the team asked whether DoriVac vaccines could also boost immune responses produced by small “neoantigens” emerging in melanoma tumors. Neoantigens are ideal targets because they are exclusively made by tumor cells. However, they often are not very immunogenic themselves, which make highly effective adjuvants an important component in neoantigen vaccines. A DoriVac vaccine customized with four neoantigens enabled the researchers to significantly suppress growth of the tumor in mice that produced the neoantigens.

Finally, the researchers asked whether DoriVac could synergize with immune checkpoint therapy, which reactivates T cells that have been silenced in tumors. In mice, the two therapies combined resulted in the total regression of melanoma tumors, and prevented them from growing back when the animals were exposed to the same tumor cells again four months later. The animals had built up an immune memory of the tumor. The team obtained a similar vaccination efficiency in a mouse lymphoma model.

We think that DoriVac’s value for determining a sweet spot in adjuvant delivery and enhancing the delivery and effects of coupled antigens can pave the way to more effective clinical cancer vaccines for use in patients with a variety of cancers.”

Yang (Claire) Zeng, M.D., Ph.D., First Author

The team is currently translating the DoriVac platform toward its clinical application, which is supported by the study’s assessment of vaccine distribution and vaccine-directed antibodies in mice, as well as cytokines produced by immune cells in response to the vaccines in vivo.

“The DoriVac platform is our first example of how our pursuit of what we call Molecular Robotics – synthetic bioinspired molecules that have programmable shape and function – can lead to entirely new and powerful therapeutics. This technology opens an entirely new path for development of designer vaccines with properties tailored to meet specific clinical challenges. We hope to see its rapid translation into the clinic,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at HMS and Boston Children’s Hospital, and the Hansjörg Wyss Professor of Bioinspired Engineering at SEAS.

Other authors on the study are Olivia Young, Christopher Wintersinger, Frances Anastassacos, James MacDonald, Giorgia Isinelli, Maxence Dellacherie, Miguel Sobral, Haiqing Bai, Amanda Graveline, Andyna Vernet, Melinda Sanchez, Kathleen Mulligan, Youngjin Choi, Thomas Ferrante, Derin Keskin, Geoffrey Fell, Donna Neuberg, Cathrine Wu, and Ick Chan Kwon. The study was funded by the Wyss Institute’s Validation Project and Institute Project programs, Claudia Adams Barr Program at DFCI, Korean Fund for Regenerative Medicine (award #21A0504L1), Intramural Research Program of KIST (award #2E30840), and National Institutes of Health (under the i3 Center supporting U54 grant (award #CA244726-01).

Source:

Wyss Institute at Harvard University

Journal reference:

Zeng, Y. C., et al. (2024). Fine tuning of CpG spatial distribution with DNA origami for improved cancer vaccination. Nature Nanotechnology. doi.org/10.1038/s41565-024-01615-3.

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March 15, 2024
Innovative CAR T cell therapy targets two proteins to combat aggressive brain tumor growth

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In a recent report published in Nature Medicine, researchers presented the initial results of recurrent glioblastoma (rGBM) patients treated with intrathecally administered, autologous, bivalent chimeric antigen receptor (CAR) T cells targeting epidermal growth factor receptor (EGFR) and interleukin-13 receptor alpha 2 (IL13Rα2) in a phase 1 clinical trial.

Study: Intrathecal bivalent CAR T cells targeting EGFR and IL13Rα2 in recurrent glioblastoma: phase 1 trial interim results. Image Credit: Gorodenkoff/Shutterstock.com

Background

Recurrent GBM is an aggressive, treatment-resistant brain cancer with no conventional therapeutic options after chemoradiotherapy. The median overall survival (OS) is less than one year, indicating that effective treatment is an urgent unmet medical need in oncology.

Despite limited therapy options, chimeric antigen receptor T cells that target GBM-specific antigens have demonstrated tolerable safety but poor effectiveness in adults.

Tumor antigen heterogeneity, intrinsic T cell malfunction, and immunosuppressive tumor microenvironments are all examples of resistance mechanisms.

About the report

In the present report, researchers describe CART-EGFR-IL13Rα2 cell efficacy and safety in treating rGBM patients recruited in an ongoing, open-label, phase 1 trial.

The researchers included six adults presenting with multifocal and progressive wild-type glioblastoma recurring after radiotherapy, excluding those who received bevacizumab within three months before trial initiation and those with localized central nervous system tumors.

They used pre-treatment immunofluorescence analysis to detect EGFR and IL13Rα2 in brain samples. The data cutoff date was February 2, 2024, with a median follow-up of 2.5 months.

Three patients received 1 × 107 cells (dose level 1), while the others received 2.5 × 107 cells (dose level 2) 17–35 days following surgery between June 14, 2023, and January 2, 2024. The primary objectives were safety, maximum tolerated dosage, dose-limiting toxicity (DLT), and adverse events.

Secondary objectives included the proportion of treated patients, manufacturing failures, objective response rate (ORR), response length, overall survival (OS), and progression-free survival (PFS).

The team obtained brain magnetic resonance imaging (MRI) images 24 to 48 hours after CAR T therapy, four weeks later, and monthly afterward.

They collected cerebrospinal fluid (CSF) at baseline and on day one, day four, day seven, day 10, day 14, day 21, and day 28 for pharmacokinetic evaluation by quantitative polymerase chain reaction (qPCR).

They rated cytokine release syndrome (CRS) according to the American Society for Transplantation and Cellular Therapy (ASTCT) guidelines and neurotoxicity using the immune effector-associated neurotoxicity syndrome (ICANS) criteria.

They assessed treatment response using the Modified Response Assessment in Neuro-Oncology (mRANO) standards.

Results

The use of CART-EGFR-IL13Rα2 cells in cancer patients was associated with early-onset neurotoxicity, perhaps ICANS. All six patients showed reduced tumor size and enhancement, but none satisfied the ORR criteria. The researchers identified CAR T cell abundance and cytokine release in all patients and all developed neurotoxicity.

Patient 1 was diagnosed with grade 2 neurotoxicity following CAR treatment, resulting in disorientation, nausea, and aphasia. Anakinra and dexamethasone improved his neurological condition.

The researchers surgically removed a tumor nodule, revealing therapy-related alterations and uncommon glial cells. After two months, the disease progressed, for which the patient received bevacizumab and continues to remain alive with an eight-month OS.

Patient 2 had rapid tumor development and facial paralysis, which indicated grade 3 neurotoxicity. The team administered anakinra and dexamethasone as treatments. On day two, the tumor mass decreased without intervention, showing pseudo-progression. The patient developed hydrocephalus symptoms, denied shunting, and died five months after receiving CAR T cell therapy.

Patient 3, presenting with deteriorating leptomeningeal illness and decreased performance, developed grade 3 neurotoxicity and received anakinra and dexamethasone treatment.

Despite fluctuations in orientation and alertness, the patient recovered on day four, reverted to his pre-treatment neurological baseline within a week, and continues to have stable disease.

Patient 4 experienced significant neurotoxicity and received dexamethasone, anakinra, and tocilizumab, resulting in a better mental state and a restoration to the pre-treatment neurological baseline. Patient 4’s enhancement foci and periventricular nodules were decreased at treatment level 2.0 and remained stable.

Patient 5 developed multifocal tumor progression and grade 2 CAR neurotoxicity, which improved to grade 1 on the third day. He developed increased weariness, physical weakness, and anorexia. The tumor burden decreased dramatically following a doubling of dexamethasone dosage.

However, the patient returned with more lethargy, fatigue, and inadequate oral intake. On day 28, raising the steroid dosage reduced the severity of multifocal irregular enhancement.

Following a CAR T cellular injection, Patient 6 had tumor development in the left midbrain and severe right-sided hemiparesis. He experienced deteriorating aphasia and increased right-sided weakness, resulting in total hemiplegia.

Despite treatment with dexamethasone and anakinra, extensive hemiparesis remained. He got bevacizumab intravenously as part of his therapy.

The first-in-human data demonstrates the safety and bioactivity of CART-EGFR-IL13Rα2 treatment in individuals with multifocal, treatment-resistant rGBM.

The therapy decreased tumor size and enhancement but resulted in early-onset acute neurotoxicity, controllable at both dosages. The findings require confirmation with larger sample sizes and longer follow-ups.

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March 15, 2024
Man takes 217 COVID vaccines with no ill effects, shows immune boost

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In a recent case report published in The Lancet Infectious Diseases, researchers described a case of a 62-year-old male who received 217 vaccinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 29 months and examined his immunological responses. They found that hyper-vaccination did not cause adverse events or significantly affect the quality of adaptive immune responses while resulting in increased T-cells and spike-specific antibodies.

Study: Adaptive immune responses are larger and functionally preserved in a hypervaccinated individual. Image Credit: Douglas Sacha / Shutterstock

Background

Booster vaccinations may potentially amplify immune responses, while persistent antigen exposure may induce immune tolerance. However, the advantages, constraints, and risks of recurrent vaccination in humans remain to be thoroughly investigated. In the present study, researchers investigated the immunological responses in an older man hyper-vaccinated against SARS-CoV-2.

The case

In this case study, a 62-year-old male from Magdeburg, Germany (referred to as HIM), engaged in deliberate hyper-vaccination against SARS-CoV-2, receiving 217 vaccinations over 29 months for personal reasons. This occurred outside a clinical study context and contrary to national recommendations. Despite an investigation by a public prosecutor for potential fraud, no criminal charges were filed. Notably, HIM’s immunological evaluation, initiated during the public prosecutor’s investigation, received active and voluntary cooperation from HIM and was ethically approved. Throughout the extensive hyper-vaccination, HIM reported no vaccine-related side effects, and routine clinical chemistry parameters displayed no abnormalities between November 2019 and October 2023. In the repeated negative SARS-CoV-2 tests, including antigen tests, polymerase chain reaction (PCR) test, and nucleocapsid serology, HIM showed no signs of past SARS-CoV-2 infection.

Starting from the 214^th vaccination, HIM’s anti-spike SARS-CoV-2 immunoglobulin G (IgG) levels were measured before and after vaccinations. The antibody peak occurred at the 214^th vaccination, and there was a slight increase after the 217^th vaccination. Additionally, HIM showed IgG4 subclass switching after the 215^th vaccination, which is uncommon in regimens with adenoviral-based vaccines as the first dose.

A total of 29 individuals who received three doses of a messenger ribonucleic acid (mRNA) vaccine formed the control group. As compared to controls, HIM exhibited mildly elevated levels of anti-spike IgM and IgA in the serum. However, in saliva samples, HIM showed detectable levels of anti-spike IgG, contrary to the control participants. HIM’s serum neutralization capacity was higher (5.4-fold for wildtype and 11.5-fold for Omicron B1.1.529 spike proteins) than the controls, indicating elevated quantities of spike-specific IgG. This observed difference was not attributed to antibody avidity as it remained comparable among the groups.

HIM showed a slightly increased number of spike-specific B-cells, with the same phenotype as seen in single-cell RNA sequencing (scRNA-seq). No significant differences were observed in the rates of somatic hypermutation or clonal expansion. CD8+ T-cells specific to the spike epitope were about six-fold more frequent in HIM, with a preference for effector memory T-cells. Further, scRNA-seq of LTD-specific T-cells showed a more differentiated phenotype and increased clonal expansion compared to controls. Flow-cytometric analysis and metabolic profiling showed no significant abnormalities in 14 protein markers.

LTD-specific CD8+ T-cells in HIM showed a proliferative capacity similar to control individuals, aligned with conserved numbers of T-cells with a phenotype like early differentiated stem cells. After epitope-specific stimulation, HIM displayed higher cytokine-positive cells, but the cytokine release per cell remained roughly equal. Cytokine analysis in the supernatant revealed the typical pattern of virus-specific CD8+ T-cells. Additionally, HIM’s CD8+ T-cells showed higher peptide sensitivity than the control group. Examination of spike-reactive CD4+ T-cells revealed a dearth of nucleocapsid-specific immunity, with similar cytokine-producing CD4+ T-cell amounts in HIM compared to the control group while retaining peptide sensitivity.

Conclusion

In conclusion, the present case report showed that hyper-vaccination against SARS-CoV-2 yielded no adverse events and elevated T-cell levels and spike-specific antibodies. Notably, the implicit quality of adaptive immune responses showed no significant effects. Although breakthrough SARS-CoV-2 infections were not observed in the individual, any causal link with the hyper-vaccination regimen remains unclear. The researchers emphasize that they do not advocate for hyper-vaccination as an approach to improve adaptive immunity.

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March 8, 2024
Study uncovers unexpected level of complexity hidden within neutrophils

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In a study published in Cell on March 5, Prof. Zhang Xiaoming at the Shanghai Institute of Immunity and Infection (SIII) of the Chinese Academy of Sciences and Profs. Gao Qiang, Fan Jia and Yang Li at Fudan University have uncovered an unexpected level of complexity hidden within neutrophils, which were previously thought to be a relatively uniform population of short-lived immune cells.

Using cutting-edge single-cell RNA sequencing technology, the researchers analyzed individual neutrophils across a remarkable 17 different cancer types from 143 patients. They revealed that neutrophils can adopt at least 10 highly specialized and distinct functional states related to inflammation, blood vessel formation, and-;most excitingly-;presenting antigens to activate potent cancer-killing T cells.

We were surprised to find such intricate complexity and divergent roles embedded within neutrophils, which have been overlooked for so long as a simple population. What is especially remarkable is their newly discovered capacity to act as antigen-presenting cells, maturing and rallying T cells against cancer. In addition, the abundance of antigen-presenting neutrophils is associated with improved patient prognosis across many tumor types revealed in this study.”

Prof. Zhang Xiaoming, Shanghai Institute of Immunity and Infection (SIII) of the Chinese Academy of Sciences

Through extensive analysis, the researchers determined that this antigen-presenting state can be switched on through metabolic signaling of the amino acid leucine and ensuing epigenetic changes. “We’ve uncovered a way to wake up an untapped army already living within our immune system. Strategically activating these neutrophil states or modulating their behavior through metabolic or dietary means represents an entirely new paradigm to empower cancer immunotherapy,” said Prof. ZHANG.

The researchers then validated the therapeutic potential of these findings through in vivo models. They found that delivering antigen-presenting neutrophils or simply modulating the leucine diet dramatically boosted the anti-tumor immune response in mice, and the treatments also markedly improved outcomes of PD-1 checkpoint immunotherapy across a range of cancer types.

“This completely changes how we perceive neutrophils in the context of cancer,” said Prof. GAO. “Now we know we could harness the diverse hidden identities of neutrophils to strengthen the effectiveness of immunotherapies. We’re thrilled to further explore the potential benefits of these newly uncovered mechanisms in clinics.”

This study underscores the value of single-cell sequencing approaches to reveal new functional dimensions even within seemingly well-understood immune cells. Tapping into the hidden potential of neutrophils may provide new insights into cancer diagnosis and treatment.

Source:

Chinese Academy of Sciences Headquarters

Journal reference:

Wu, Y., et al. (2024). Neutrophil profiling illuminates anti-tumor antigen-presenting potency. Cell. doi.org/10.1016/j.cell.2024.02.005.

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March 7, 2024
What factors are associated with recent prostate-specific antigen screening in transgender women?

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A new report in JAMA Network Open reports on factors associated with prostate cancer screening among transgender women.

Study: Prostate Cancer Screening Uptake in Transgender Women. Image Credit: Jarun Ontakrai/Shutterstock.com

Background

Not much is known about the incidence of prostate cancer in this population, but some research indicates that it may be as high as 14 per 10,000 cases. Moreover, transgender women on hormone therapy are more likely to develop aggressive disease, potentially due to diagnostic delay from misinterpreting the prostate-specific antigen (PSA) test values in the context of the suppressive effects of estrogen.

As of now, there is no guideline on when prostate cancer screening via (PSA) test should be done in transgender women. However, it is known that they are at increased risk for this cancer since prostatectomies are not part of the surgical procedure to change gender in such individuals.

The current study sought to identify factors common to transgender women who underwent a PSA screening test during the previous two years compared to cisgender men.

What did the study show?

The data came from the Behavioral Risk Factor Surveillance System (BRFSS) surveys of 2018 and 2020, which were carried out by the Centers for Disease Control and Prevention (CDC). This survey covers more than 400,000 American adults each year.

In the current study, there were nearly 1,300 participants, all 40 years or above, without a history of prostate cancer. Approximately 260 transgender women were matched to over 1,000 men. About 45% were between 55 and 69 years old. Almost 80% were White.

Transgender women were less likely to have a college degree or have current work and found it more difficult to pay healthcare costs compared to cisgender men. The odds of their earning $75,000 or more yearly were much lower compared to cisgender men.

In the age group of 55-69 years, PSA screening was performed within the previous two years in over one in five transgender women vs well over one in three cisgender men. The percentages in the over-70 group were similar between the groups, at ~40%.

When matched for confounding factors, such as whether a doctor had recommended or discussed a PSA test or time since the last clinical visit, there was no significant difference in screening proportions between the two groups. The strongest factor associated with PSA screening among transgender women was a doctor recommending the test, with such individuals being over 12 times more likely to be tested. Similarly, if a doctor had discussed the advantages of PSA testing, the odds were almost 8-fold.

However, those with higher educational status and higher incomes were also more likely to have been recently screened. Having a college degree increased the odds of recent screening by 2.55 times.

Older individuals (70 years or more) were almost twice as likely to have been screened recently vs those aged 55-69 years. This corroborates earlier reports. The difference in screening rates in the younger age group remained unexplained and did not vary with primary care follow-up frequency.

What are the implications?

This was among the largest cohort studies of PSA screening among transgender women so far. While it appeared on the surface that transgender women were less likely to undergo screening than cisgender men, the difference became smaller and statistically insignificant when accounting for the influence of a doctor’s recommendation to have the test done.

The large effect of clinician recommendations on the rate of PSA screening among transgender women makes it important that doctors educate themselves on how best to treat transgender patients. Further study is essential to arrive at a quantitative estimate of the cost-effectiveness of PSA screening among transgender women.

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February 19, 2024
New study pinpoints key markers for Long COVID diagnosis
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In a recent preprint* uploaded to the medRxiv server, an international team of researchers conducted a large-scale systems-level immunological screening of more than 1,000 confirmed COVID-19 patients to identify diagnostic markers of Long-term COVID-19. The analyses using multiple orthogonal detection methods reveal elevated serologic responses as a highlight of Long COVID and that its correlated memory CD8⁺ T cell clonal expansion is a more reliable and sensitive marker of the condition than conventional antigen (SARS-CoV-2 RNA and protein) detection approaches.

Study: Restrained memory CD8⁺ T cell responses favors viral persistence and elevated IgG responses in patients with severe Long COVID. Image Credit: Lightspring / Shutterstock

*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

COVID-19 and the need for Long COVID diagnosis

The ongoing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused Coronavirus disease 2019 (COVID-19) viral pandemic is one of the worst in human memory, estimated to have infected more than 700 million individuals since its discovery in Wuhan, China, in late 2019. While global legislative policy and the widespread development and dissemination of anti-COVID-19 vaccines have substantially reduced the disease burden, with reports of vaccination efforts saving 70% of patients or more, survivors of the pandemic are plagued by a hitherto unknown condition – Long COVID.

Also called the ‘Post COVID-19 condition’, ‘chronic COVID syndrome,’ and clinically, ‘post-acute sequelae of COVID-19 (PASC)’, Long COVID presents itself as perhaps the worst legacy of the pandemic. The now well-established yet poorly understood condition is characterized by the persistence or development of COVID-19-associated symptoms that may persist for months or even years following initial infection recovery. These symptoms include severe cognitive decline (brain fog), chronic fatigue, and multiple organ damage, resulting in significant economic and quality of life (QoL) losses in patients.

Alarmingly, research has revealed that despite vaccination efforts substantially reducing adverse Long COVID outcomes, between 30 and 60% of all COVID-19 infections result in Long COVID, with an estimated 350+ million individuals suffering from the condition. Unfortunately, extensive global scientific efforts remain unable to elucidate the mechanisms underpinning Long COVID, hampering the development of diagnostic assays and clinical interventions for patients.

About the study

In the present study, researchers screened more than 1000 prospective patients enrolled at Long COVID clinics in Belgium and Sweden to elucidate the shared mechanisms of Long COVID pathology and subsequently develop a sensitive and reliable diagnostic test for the condition. Only subjects with a clinically confirmed mild or moderate COVID-19 infection were included. Severe cases were excluded due to overlapping symptoms with those of the post-intensive care syndrome.

Patients without objective measures of disease-associated organ damage (e.g., magnetic resonance imaging [MRI], pulsatile arterial tonometry [EndoPAT], and postural orthostatic tachycardia syndrome [POTS]) were excluded. Inclusion and exclusion criteria resulted in a final sample cohort of 121 patients from Belgium (n = 31) and Sweden (n = 90).

Experimental procedures included the enzyme-linked immunosorbent assay (ELISA) for detecting and measuring patients’ antibody responses against SARS-CoV-2. Since these standard ELISAs were not observed to elucidate differences in immunoglobulin A (IgA) and IgM despite clear case-convalescent control differences in IgG titers, single-molecule array (SIMOA) assays were employed. The SPEAR immunoassay was used to detect the presence of persistent SARS-CoV-2 spike proteins in patients’ plasma samples.

Since these assays revealed that antigen responses were only depicted by about 10% of the study cohort, suggesting its unreliability and poor sensitivity as a diagnostic tool, researchers used a 51-parameter-panel mass cytometry assay to investigate possible immunological correlates. The Olinks assay was further conducted to measure levels of cytokines and other plasma proteins in patients’ plasma samples.

“Autoantibodies to type-I IFN have been associated with life-threatening COVID-19 pneumonia due to impaired IFN-I-mediated inhibition of viral replication. Such autoantibodies increase in frequency with age, are more common in males than females for unknown reason, and could explain up to 20% of COVID-19 deaths. The reasons for the development of anti-cytokine autoantibodies are unknown in most cases, but most, if not all, patients with inborn errors of central tolerance due to AIRE deficiency in cis (APECED or APS1) or in trans (mutations of the alternative NF-kB pathway) all carry these autoantibodies and are highly susceptible to severe SARSCoV-2 infections.”

To investigate the above, single-cell T-cell receptor (TCR) and message RNA (mRNA) sequencing of peripheral blood mononuclear cells (PBMCs) was carried out. Memory CD8 T cell TCR sequences were then clustered using the GLIPH methodology.

Study findings

The present study reveals that, while IgG response to SARS-CoV-2 spike (receptor binding domain [RDB]) proteins as measured by the SIMAO assay can be used as a sensitive Long COVID marker, IgA and IgM cannot due to their detection in ~10% of afflicted patients. This suggests that memory CD8⁺ T cells were restrained, and their clonal expansion is restricted by SARS-CoV-2, inconsistent with the previously hypothesized exhausted phenotype pathology.

Strong and persistent Long COVID symptoms despite high IgG tirtes suggest differences between the initial and long-term adaptive responses of patients’ immunity to SARS-CoV-2.

“A strong initial adaptive response might increase the chance of viral clearance and reduce the risk of Long COVID, while a sustained and elevated long-term response to SARSCoV-2 with elevated titers occur once a viral reservoir has been established leading to chronic antigen stimulation.”

Results highlight that in Long COVID cases, the elevated serologic response was inversely correlated to expanding CD8⁺T cell populations, elucidating the role of the restrained antiviral T cell response as a crucial component of Long COVID pathology. Current and future work aimed at understanding the genetic basis of this revelation may allow for the development of clinical therapeutics capable of treating this hitherto incurable condition.

Conclusions

The present study uses a combination of ELISA, SIMOA, and sequencing assays to investigate the associations between circulating immunoglobulin titers and Long COVID pathology, with the dual aim of elucidating Long COVID’s mechanism of action and progressing the discovery of a universal Long COVID diagnostic test. Their findings reveal that contrary to expectation, IgG titers in Long COVID patients increase following initial infection recovery, suggesting chronic antigen stimulation.

IgA and IgM titers, in contrast, were extremely low and detectable in only 10% of cases, making them unreliable in Long COVID diagnosis.

*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
Journal reference:

Preliminary scientific report.
Lucie Rodriguez, Ziyang Tan, Lakshmi Kanth Tadepally, Jun Wang, Hugo Barcenilla, Zoe Swank, Fanglei Zuo, Hassan Abolhassani, Ana Jimena Pavlovitch-Bedzyk, Chunlin Wang, Laura Gonzalez, Constantin Habimana Mugabo, Anette Johnsson, Yang Chen, Anna James, Jaromir Mikes, Linn Kleberg, Christopher Sundling, Mikael Bjornson, Malin Nygren-Bonnier, Marcus Stahlberg, MIchael Runold, Sofia Bjorkander, Erik Melen, Isabelle Meyts, Johan Van Weyenbergh, Qiang Pan Hammarstrom, Mark M Davis, David R. Walt, Nils Landegren, COVID Human Genetic Effort, Alessandro Aiuti, Giorgio Casari, Jean-Laurent Casanova, MARC JAMOULLE, Judith Bruchfeld, Petter Brodin. Restrained memory CD8+ T cell responses favors viral persistence and elevated IgG responses in patients with severe Long COVID. medRxiv (2024), DOI – 10.1101/2024.02.11.24302636, https://www.medrxiv.org/content/10.1101/2024.02.11.24302636v1
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February 18, 2024
Study suggests patients with severe long COVID present with variable symptoms, do not cluster in relation to organs affected or immunological states

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A new preprint, recently uploaded to the medRxiv* preprint server, reports significantly associated findings that may help predict severe long COVID and understand what causes it.

Study: Restrained memory CD8+ T cell responses favors viral persistence and elevated IgG responses in patients with severe Long COVID. Image Credit: Anucha Naisuntorn/Shutterstock.com

*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Background

SARS-CoV-2 infection causes protean manifestations extending over a wide range of severity. In the most severe cases, multiorgan failure and death supervene, with underlying coagulation failure and hyperinflammation. The key factor is the occurrence of an antiviral type-1 interferon (IFN-1) response to infection of respiratory epithelial cells and antigen-presenting dendritic cells, as this is responsible for further events that limit viral replication. In addition, it allows immune clearance of the virus.

A type of post-COVID sequel among children has been termed the Multisystem Inflammatory Syndrome in Children (MIS-C). The occurrence of this condition is associated with the persistence of the virus in the gut, perhaps linked to chronic T cell activation.

The resemblance to superantigen-induced T cell activation did not escape notice, especially as a superantigen-like motif had already been reported in the SARS-CoV-2 spike protein with similarity to staphylococcal enterotoxin B. Coupled with potential genetic defects associated with MIS-C, the authors speculate a common origin for both long COVID and MIS-C.

According to them, “disease tolerance, restrained antiviral T cell responses and viral persistence” comprise one such possibility. They believe that in select groups, such as children and women of reproductive age, efficient antiviral responses ensure mild infections by limiting systemic inflammation and T cell responses. On the flip side, this increases the odds of viral persistence and, thus of long COVID.

Evidence for viral persistence is scanty, including the presence of viral antigens in blood and tissue samples and imaging reports, as well as finding continued somatic hypermutation in B cells specific to this virus. Again, vaccination against COVID-19 reduces the risk of long COVID, as does early antiviral therapy, supporting the hypothesis that this sequel results from viral persistence.

The current report centers on patients who had mild to moderate COVID-19 and later developed severe long COVID.

What did the study show?

The scientists examined only patients with documented mild to moderate SARS-CoV-2 infection who had not been hospitalized over a thousand of them. From these, they selected those who had evidence of organ damage to the heart, blood vessels, autonomic nervous system, hyperventilation, or changes in computerized tomography (CT) of the lungs.

These were chosen since they provided objective proof of organ damage, supporting the diagnosis of severe long COVID. They found ~120 cases, almost 90% being females with the mean age being 48 years.

They found that there is no common set of symptoms, signs of organ damage, or characteristic immunological profile in patients who develop long COVID. However, serum antibody responses were markedly higher in these patients compared to those who recovered rapidly. This is a sign of persistent antigen stimulation, especially as these cases were sampled long after the acute infection.

When viral RNA and protein antigens were looked for in plasma samples, using an array of independent testing methods, the researchers found that only a subset of patients were positive for viral antigens by any of the methods used. There was little overlap in the results between different assays. This indicates that blood tests for viral persistence may not yield uniform results, and the finding of elevated antibody levels is a more sensitive marker of long COVID.

The failure to detect antigens by all assays could either reflect the decreased sensitivity of the tests or the fact that the virus harbored in the tissue reservoirs may not leak the antigens into the bloodstream. It is unlikely that failure of viral persistence is the reason, given the persistent increase in anti-SARS-CoV-2 IgG responses observed in multiple cohorts.

They also found certain monocyte subsets and plasma proteins associated with an ongoing innate immune response at elevated levels. Such elevations were in proportion to increased immunoglobulin G (IgG) antibody titers, a marker of persistent inflammation.

In contrast, there was a failure of expansion of cytotoxic memory CD8+ T cells targeting SARS-CoV-2. The frequency of such clones decreases as antibody levels rise.

“This suggests that individuals who fail to mount a clonally expanded memory CD8+ T cell response to SARS-CoV-2 develop a viral reservoir with persistent antigen.”

The researchers did not find autoantibodies to IFN-1 in these patients. However, these have been found in life-threatening COVID-19-associated pneumonia and account at least in part for one in five deaths due to COVID-19 by inhibiting IFN-1-mediated suppression of viral replication.

This indicates that the underlying mechanism of long COVID is viral persistence, with impaired CD8+ T cell responses being the norm, rather than T cell exhaustion, as some scientists have suggested earlier to be characteristic of severe long COVID.

What are the implications?

The study suggests that the finding of elevated SARS-CoV-2-specific IgG sensitively identifies long COVID mounted against the spike antigen. Secondly, this is likely to be the result of viral persistence with chronic antigen stimulation.

Those patients who respond strongly to the initial infection via an adaptive immune response likely clear the virus rapidly and have a lower chance of long COVID. In contrast, if a viral reservoir is established, chronic stimulation by viral antigens leads to a long-term elevation of anti-SARS-CoV-2 IgG.

The researchers continue to seek the reason for the failure of viral clearance in some patients leading to long COVID. Until then, prolonged administration of antivirals may be the best way to treat these patients by removing the viral reservoir.

*Important notice: medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

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February 16, 2024