Tag: CD4

New treatment rejuvenates elderly defenses
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In a recent study published in Nature, researchers developed a treatment to restore the immunological system to a younger condition, with fewer myeloid-biased output-hematopoietic stem cells (my-HSCs), more HSCs, and a balanced generation of myeloid and lymphoid lineage cells (bal-HSCs).

Study: Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity. Image Credit: Lightspring / Shutterstock.com

The effects of aging on the immune system

The aging immune system is associated with reduced lymphopoiesis, increased inflammation, and myeloid diseases due to alterations in self-renewing HSCs. During childhood, bal-HSCs predominate, thereby facilitating lymphopoiesis and adaptive immune responses.

Age increases my-HSCs, which reduces lymphopoiesis and enhances myelopoiesis. Myeloid-HSC origin and possible interconversions are unclear; however, removing my-HSCs in aged mice may reverse the aging phenotype.

About the study

The researchers investigated whether antibody-regulated reduction of my-HSCs may cure age-related immunological reductions by restricting myeloid cell-induced inflammation and restoring lymphopoiesis. To this end, the impact of reduced my-HSCs on the hematopoietic system, immunological phenotypes, and functional responses to incident infections was assessed.

Several cell-surface antigen molecules were developed and validated to identify potential targets for therapeutic my-HSC reduction. The levels of my-HSCs and balanced-HSCs were determined using antibodies and flow cytometry.

Several my-HSC antigens, including neogenin 1 (NEO1), cluster of differentiation 62p (CD62p), and CD150, were subsequently targeted to determine their role in reducing my-HSC levels. Separate antibody-conditioning treatments were then developed for my-HSC depletion for each target, with a focus on cell clearance regulators such as anti-phagocytic signals, isotype, and antibody density.

To establish the role of CD150 targeting, the ability of CD150-targeted antibodies to reduce my-HSCs in vivo was assessed. To target CD62p or NEO1, goat anti-mouse NEO1 antisera was mixed with anti-CD47 and anti-KIT antibodies.

Gene expression analysis of pure total HSCs extracted from 11-month-old mice was performed to confirm alterations in HSC composition following my-HSC elimination. Transplant tests using pure HSCs were also performed to compare the myeloid and lymphoid lineage potential in recipient mice.
After antibody conditioning, myeloid and common lymphocyte progenitors (CLPs) were measured in murine bone marrow. These analyses were performed after one week to assess acute effects, as well as after eight and sixteen weeks to determine long-term effects. The impact of this treatment on non-self-renewing progenitors was also evaluated after eight weeks.

T-cell subsets were analyzed using canonical markers or cluster-based analysis. The effects of my-HSC depletion in aged animals on pro-inflammatory mediators and functional immunity to infection were also examined by analyzing mouse immune responses to a live-attenuated virus and subsequent challenge with a pathogenic viral infection using the mouse Friend retrovirus (FV) model.

Study findings

Antibody-mediated reduction of my-HSCs in elderly mice restored young immune system characteristics, such as increased CLPs, naïve T-cells, and B-cells, while lowering immunological decline indicators associated with aging. Depletion of my-HSCs in old mice increased primary and secondary adaptive immune responses to viral infection.

Twelve potential genes that encode cell-surface proteins significantly expressed in aged HSCs and my-HSCs were identified. Moreover, CD150, CD4, CD6, CD62p20, and NEO1 were identified as markers for my-HSCs.

Antibodies to CD41 and NEO1 enhanced the frequency of my-HSC staining, thus indicating myeloid bias. CD62p targeting resulted in the highest my-HSC enrichment.

The most abundant protein molecules on my-HSCs were NEO1, CD41, and CD62p. Flow cytometry analysis did not identify any surface protein strongly expressed by the subgroups, except CD41, which was highly expressed by megakaryocyte progenitor cells.

Anti-CD150 antibodies significantly reduced my-HSCs in mice, thereby increasing naïve T-cell and mature B-cell levels. In aged mice, CD4+ T lymphocytes with an exhausted phenotype (PD1+ CD62L-) grew more than those with a non-exhausted phenotype (PD1- CD62L+).

Antibody training reduced CD4+ PD1+ CD62L- cells as compared to CD4+ PD1- CD62L+. Aged mice also acquired age-associated B-cells associated with impaired humoral immunity.

Antibody training reduced the levels of pro-inflammatory proteins including interleukin-1 alpha (IL-1α), and C‐X‐C motif chemokine ligand 5 (CXCL5), which were higher in elderly animals. Aged animals with my-HSC depletion exhibited higher virus-specific CD8+ T-cell responses in the spleen following vaccination, thus indicating a better initial response to live-attenuated viral infection.

Conclusions

Rising my-HSC levels during aging may result in inadequate adaptive immunological and inflammatory responses. Thus, depleting my-HSCs may improve immune responses by enhancing the synthesis of new T- and B-cells while decreasing the production of inflammatory myeloid cells. In the current study, my-HSC depletion in older animals allowed bal-HSCs to recover youthful immunological characteristics such as enhanced lymphocyte progenitors and naïve cells and decreased lymphocyte dysfunction or exhaustion indicators and inflammatory mediators.

Further research could refine conditioning techniques and examine the impact on differentiated cells, such as regulatory T-cells.
Journal reference:

Ross, J. B., Myers, L. M., Noh, J. J., et al. (2024). Depleting myeloid-biased haematopoietic stem cells rejuvenates aged immunity. Nature. doi:10.1038/s41586-024-07238-x
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April 1, 2024
New insights into the exacerbation of psoriasis through specific genetic defects

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In a recent study featured in Nature Communications, researchers created mice that carry a gain-of-function (GoF) mutation in the gene encoding the inhibitor of nuclear factor kappa-b kinase subunit beta (IKBKB), known as the IKK2-encoding IKBKB gene. This was done to explore how this mutation works.

Study: IKK2 controls the inflammatory potential of tissue-resident regulatory T cells in a murine gain of function model. Image Credit: Gorodenkoff/Shutterstock.com

Background

Loss-of-function mutations demonstrate the importance of forkhead box P3-positive (Foxp3+) regulatory T cells (Tregs) in immunological control. Tregs mediate dominant tolerance and protect against autoimmune disorders.

They undergo positive selection in the thymus, and interleukin-2 (IL-2) protects them from apoptosis. Treg formation needs effective signaling downstream of the T-cell receptor (TCR), particularly the CARD11-BCL10-MALT1 (CBM) complex.

Mice lacking particular genes have a Treg deficit that causes a selective loss of cluster of differentiation 4-positive (CD4+) Helios+ thymic T cells.

Tregs move between lymphoid organs according to adhesion molecule expression. The presence of an activated or effector phenotype (eTreg) in recirculating Tregs increases disease risk.

About the study

The present study examined mice with an Ikbkb GoF mutation homologous to a problematic human IKBKB GoF variation.

The researchers tracked a cohort of mice with various Ikbkb genotypes and recorded the age at which skin disease appeared. Animal house technicians were unaware of the mouse genotype and identified abnormal Ikbkbmut/+ and Ikbkbmut/mut animals. The researchers examined the transcriptomes of tails and ears from Ikbkbmut/mut and Ikbkb+/+ mice.

The team investigated the inflammatory infiltrate in skin lesions and the nature of Treg growth inside pathological lesions. They created mixed bone marrow chimeras with allotype-marked donor cells from WT and mutant mice.

They isolated naïve CD4+ T cells from mouse splenocyte suspensions and activated them with Th17-inducing conditions. The researchers then counted IL-17+ Tregs ex vivo and labeled them for cytokine production after gating on Foxp3.

The researchers extracted them from WT mice and cocultured them with pure WT conventional T cells labeled with CTV to explore Tregs’ traditional immunosuppressive activity. They followed up with an in vivo test of mutant Treg suppression.

They analyzed mice for signs of systemic immunological dysregulation and created reciprocal bone marrow (BM) chimeras to study Ikbkbmut’s cell-intrinsic effects on the Treg phenotype.

The team obtained serum from recipient mice to analyze a panel of cytokines. They isolated green fluorescent protein (GFP)-labeled Foxp3+ Tregs from Ikbkbmut donors and implanted them into Ikbkbmut x Rag1−/− or IkbkbWT x Rag1−/− animals to establish disease cause as pro-inflammatory Treg activity.

The researchers used mice aged six weeks to 12 months for analysis. They performed flow cytometry, flow cytometric cell sorting, ex vivo PMA/ionomycin stimulation for cytokine production, T-cell polarization, an in vitro Treg suppression experiment, cell trace violet (CTV) labeling, and single-cell and bulk ribonucleic acid (RNA) sequencing studies.

Results

Canonical NF-κB overactivity led to the growth of pathogenic, NF-κB-dependent, and modified non-lymphoid tissue skin Tregs. Mice with Ikbkb GoF mutation heterozygosity developed psoriasis, and Ikbkb-mut mice included IL-17-producing Tregs.

These animals maintained suppressive function, indicating that normal CD4+ T cells are not the source of IL-17 in Ikbkb mutant mice. Foxp3+ CD4+ T cells from Ikbkb mutant mice maintained suppressive function.

The study additionally examined the effects of doubling the IkbkbGoF/GoF gene dosage on psoriatic arthritis, characterized by spondylitis, dactylitis, and distinctive nail abnormalities.

IkbkbGoF mice showed selective CD25+ and Foxp3+ Treg expansion, with a fraction expressing IL-17. These transformed Tregs were present in inflamed tissues, spleen, and blood, and their transfer was sufficient to cause illness without ordinary T lymphocytes.

Single-cell phenotyping and transcriptional investigations of isolated regulatory T cells indicated the non-lymphocytic tissue proliferation of Treg expressing Th17-associated genes, Helios, tissue-related markers such as CD69 and CD103, and a significant nuclear factor kappa B (NF-κB) transcriptome.

Overactive IKK2 caused dermal Treg accumulation and psoriasis. Heterozygous (Ikbkbmut/+) and homozygous (Ikbkbmut/mut) mutant mice developed skin illnesses with histopathological similarities to psoriasis.

Humans heterozygous for IKBKBV203I have combined immune insufficiency, but their Treg count increased. Ikbkbmut has a similar phenotype, with gene-dose-dependent lymphopenia caused by a decrease in αβ and γδ T cells in homozygous mice.

The study also found an increase in Th17 CD4+ T cells, strongly associated with psoriasis. Ikbkbmut/mut mice spleen Tregs produced more IL-17 than wild-type mice.

Interferon-gamma (IFNγ) production by Tregs was similar between WT and mutant animals, indicating that Ikbkbmut imparts an expansion of the IL-17-producing Foxp3+ Treg population.

Foxp3 deficiency and Treg functional abnormalities were associated with early-onset and severe widespread lymphadenopathy unrelated to the Ikbkbmut mutation.

Conclusion

The study linked psoriasis and psoriatic arthritis to NF-κB malfunction, which causes non-specific leukocytes to acquire an effector-like function, resulting in disease. The primary finding is a route that leads Foxp3+CD4+ tissue-resident Tregs to turn pro-inflammatory and pathogenic.

In vivo, a modified Treg population emerges owing to enhanced activity of the canonical NF-κB pathway. This route controls Treg abundance, increases tissue-resident Tregs, and mediates end-organ pathologies.

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March 27, 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
Unlocking the secrets of T follicular helper cells for better treatment strategies

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T follicular helper cells (Tfh) are essential for strong antibody-mediated reactions of our immune system during infections and vaccinations. However, if they get out of control, this can cause diseases such as autoimmunity, allergies or cancer. Researchers from the University Hospital Bonn (UKB) and the Cluster of Excellence ImmunoSensation at the University of Bonn investigated the underlying mechanisms of Tfh cell development in a mouse model and thus decoded their internal networking. They hope that this will lead to new strategies for the development of highly effective vaccines and new therapies to combat various diseases. The results have now been published in the renowned journal Science Immunology.

T follicular helper cells (Tfh cells) are a specialized subgroup within the so-called CD4⁺ T helper cells in the immune system. Their main task is to assist the B cells in the immune defense. They are essential for the generation of highly effective antibodies. Tfh cells therefore play a decisive role in protecting against and fighting infections. “Although Tfh cells were first described over 20 years ago, there is still no reliable protocol for their generation in cell culture,” says co-first author Dr. Yinshui Chang, former postdoctoral researcher at the University of Bonn at the UKB, describing the motivation to take a closer look at the process in the mouse model.

The transforming growth factor TGF-β is a cytokine. This is a group of proteins that initiates and regulates the growth and differentiation of cells. The Bonn team led by Prof. Dr. Dirk Baumjohann has now discovered that this signaling molecule induces strong protein expression of both the transcription factor Bcl6 and the chemokine receptor CXCR5, which are characteristic of Tfh cells. The latter plays an important role in the targeted migration of Tfh cells into the vicinity of B cells.

We were able to show that the Tfh cells induced by TGF-β in cell culture are quite similar to the Tfh cells generated in a living organism. They provide crucial help for B cells.”

Luisa Bach, co-first author, doctoral student at the University of Bonn at the UKB

Transcription factor c-Maf controls the fate of T helper cells

Using a new method based on CRISPR gene scissors, the international team led by the Bonn researchers discovered that the production of CXCR5 induced by TGF-β is independent of the transcription factor Bcl6, but requires the transcription factor c-Maf. Remarkably, although Tfh and Th17 cells partially undergo common developmental stages, c-Maf acts as a switching factor for Tfh versus Th17 cell fates. Th17 cells are another special type of CD4⁺ T helper cells and play an important role in bacterial infections and autoimmune diseases.

“Overall, our data clarify important aspects of the long-unclear prerequisites and molecular pathways for the development of Tfh cells. They also highlight the diverse functions of the transforming growth factor TGF-β. Furthermore, these data indicate that Tfh cell development in mice and humans may not be as different as we previously assumed,” says Prof. Baumjohann from the Medical Clinic III for Hematology, Oncology, Immuno-Oncology and Rheumatology at the UKB, who is a member of the Cluster of Excellence ImmunoSensation and the Transdisciplinary Research Area (TRA) “Life & Health” at the University of Bonn. “Importantly, our findings may have implications for the development of new therapeutic strategies that enhance Tfh cells during vaccinations and infections or inhibit them in autoimmune and allergic diseases.”

Source:

University Hospital Bonn (UKB)

Journal reference:

Chang, Y., et al. (2024) TGF‑β specifies TFH versus TH17 cell fates of murine CD4+ T cells through c-Maf. Science Immunology. doi.org/10.1126/sciimmunol.add4818.

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March 1, 2024
New study reveals key to blocking infection

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As the HIV virus glides up outside a human cell to dock and possibly inject its deadly cargo of genetic code, there’s a spectacularly brief moment in which a tiny piece of its surface snaps open to begin the process of infection.

Seeing that structure snap open and shut in mere millionths of a second is giving Duke Human Vaccine Institute (DHVI) investigators a new handle on the surface of the virus that could lead to broadly neutralizing antibodies for an AIDS vaccine. Their findings appear Feb. 2 in Science Advances.

Being able to attach an antibody specifically to this little structure that would prevent it from popping open would be key. Their findings appear Feb. 2 in Science Advances.

The moving part is a structure called envelope glycoprotein, and AIDS researchers have been trying to figure it out for years because it is a key part of the virus’ ability to dock on a T-cell receptor known as CD4. Many parts of the envelope are constantly moving to evade the immune system, but vaccine immunogens are designed to stay relatively stable.

Everything that everybody’s done to try to stabilize this (structure) won’t work, because of what we learned. It’s not that they did something wrong; it’s just that we didn’t know it moves this way.”

Rory Henderson, lead author, structural biologist who is an associate professor of medicine in DHVI

Postdoctoral researcher and study co-author Ashley Bennett offers a play-by-play: As the virus feels for its best attachment point on a human T-cell, the host cell’s CD4 receptor is the first thing it latches onto. That connection is what then triggers the envelope structure to pop open, which in turn, exposes a co-receptor binding site “and that’s the event that actually matters.”

Once both molecules of the virus are bound to the cell membrane, the process of injecting viral RNA can begin. “If it gets inside the cell, your infection is now permanent,” Henderson said.

“If you get infected, you’ve already lost the game because it’s a retrovirus,” Bennett agrees.

The moving structure they found protects the sensitive co-receptor binding site on the virus. “It’s also a latch to keep it from springing until it’s ready to spring,” Henderson said. Keeping it latched with a specific antibody would stop the process of infection.

To see the viral parts in various states of open, closed and in-between, Bennett and Henderson used an electron accelerator at the Argonne National Laboratory outside Chicago that produces X-rays in wavelengths that can resolve something as small as a single atom. But this expensive, shared equipment is in high demand. The AIDS researchers were awarded three 120-hour blocks of time with the synchrotron to try to get as much data as they could in marathon sessions. “Basically, you just go until you can’t anymore,” Bennett said.

Earlier research elsewhere had argued that antibodies were being designed for the wrong shapes on the virus and this work shows that was probably correct.

“The question has been ‘why, when we immunize, are we getting antibodies to places that are supposed to be blocked?’” Henderson said. Part of the answer should lie in this particular structure and its shape-shifting.

“It’s the interplay between the antibody binding and what this shape is that’s really critical about the work that we did,” Henderson said. “And that led us to design an immunogen the day we got back from the first experiment. We think we know how this works.”

This research was supported by the National Institutes of Health (UM1AI14437, R01AI145687, U54AI170752, P30 GM124169, S10OD018483), the Department of Energy (DE-AC02-06CH11357) and the DOE Office of Biological and Environmental Research.

Source:

Journal reference:

Bennett, A. L., et al. (2024) Microsecond dynamics control the HIV-1 Envelope conformation. Science Advances. doi.org/10.1126/sciadv.adj0396.

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February 3, 2024