TGFß prevents IgE-mediated allergic disease by restraining T follicular helper 2 differentiation.

Allergic diseases are common, affecting more than 20% of the population. Genetic variants in the TGFß pathway are strongly associated with atopy. To interrogate the mechanisms underlying this association, we examined patients and mice with Loeys-Dietz syndrome (LDS) who harbor missense mutations in the kinase domain of TGFΒR1/2. We demonstrate that LDS mutations lead to reduced TGFß signaling and elevated total and allergen-specific IgE, despite the presence of wild-type T regulatory cells in a chimera model. Germinal center activity was enhanced in LDS and characterized by a selective increase in type 2 follicular helper T cells (TFH2). Expression of Pik3cg was increased in LDS TFH cells and associated with reduced levels of the transcriptional repressor SnoN. PI3Kγ/mTOR signaling in LDS naïve CD4+ T cells was elevated after T cell receptor cross-linking, and pharmacologic inhibition of PI3Kγ or mTOR prevented exaggerated TFH2 and antigen-specific IgE responses after oral antigen exposure in an adoptive transfer model. Naïve CD4+ T cells from nonsyndromic allergic individuals also displayed decreased TGFß signaling, suggesting that our mechanistic discoveries may be broadly relevant to allergic patients in general. Thus, TGFß plays a conserved, T cell-intrinsic, and nonredundant role in restraining TFH2 development via the PI3Kγ/mTOR pathway and thereby protects against allergic disease.

SARS-CoV-2 reservoir in post-acute sequelae of COVID-19 (PASC).

Millions of people are suffering from Long COVID or post-acute sequelae of COVID-19 (PASC). Several biological factors have emerged as potential drivers of PASC pathology. Some individuals with PASC may not fully clear the coronavirus SARS-CoV-2 after acute infection. Instead, replicating virus and/or viral RNA-potentially capable of being translated to produce viral proteins-persist in tissue as a 'reservoir'. This reservoir could modulate host immune responses or release viral proteins into the circulation. Here we review studies that have identified SARS-CoV-2 RNA/protein or immune responses indicative of a SARS-CoV-2 reservoir in PASC samples. Mechanisms by which a SARS-CoV-2 reservoir may contribute to PASC pathology, including coagulation, microbiome and neuroimmune abnormalities, are delineated. We identify research priorities to guide the further study of a SARS-CoV-2 reservoir in PASC, with the goal that clinical trials of antivirals or other therapeutics with potential to clear a SARS-CoV-2 reservoir are accelerated.

Development of an mRNA-lipid nanoparticle vaccine against Lyme disease.

Lyme disease is the most common vector-borne infectious disease in the United States, in part because a vaccine against it is not currently available for humans. We propose utilizing the lipid nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) platform to generate a Lyme disease vaccine like the successful clinical vaccines against SARS-CoV-2. Of the antigens expressed by Borrelia burgdorferi, the causative agent of Lyme disease, outer surface protein A (OspA) is the most promising candidate for vaccine development. We have designed and synthesized an OspA-encoding mRNA-LNP vaccine and compared its immunogenicity and protective efficacy to an alum-adjuvanted OspA protein subunit vaccine. OspA mRNA-LNP induced superior humoral and cell-mediated immune responses in mice after a single immunization. These potent immune responses resulted in protection against bacterial infection. Our study demonstrates that highly efficient mRNA vaccines can be developed against bacterial targets.

Germinal center responses to SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals.

Vaccine-mediated immunity often relies on the generation of protective antibodies and memory B cells, which commonly stem from germinal center (GC) reactions. An in-depth comparison of the GC responses elicited by SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals has not yet been performed due to the challenge of directly probing human lymph nodes. Herein, through a fine-needle aspiration-based approach, we profiled the immune responses to SARS-CoV-2 mRNA vaccines in lymph nodes of healthy individuals and kidney transplant recipients (KTXs). We found that, unlike healthy subjects, KTXs presented deeply blunted SARS-CoV-2-specific GC B cell responses coupled with severely hindered T follicular helper cell, SARS-CoV-2 receptor binding domain-specific memory B cell, and neutralizing antibody responses. KTXs also displayed reduced SARS-CoV-2-specific CD4 and CD8 T cell frequencies. Broadly, these data indicate impaired GC-derived immunity in immunocompromised individuals and suggest a GC origin for certain humoral and memory B cell responses following mRNA vaccination.

A combined fine needle aspiration and spectral flow cytometry approach to assess human germinal center responses to SARS-CoV-2 vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines drive the generation of affinity-matured B cell responses through germinal center (GC) reactions in vaccine draining lymph nodes. Herein, we describe a procedure for the acquisition of human lymph node samples via an ultrasound-guided fine needle aspiration-based approach. Additionally, we outline a suggested approach for the analysis of CD4 T helper cell subsets as well as antigen-specific GC B cells, memory B cells, and plasmablasts by high-parameter spectral flow cytometry. For complete details on the use and execution of this protocol, please refer to Lederer et al. (2022).1.

Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses.

Adjuvants are critical for improving the quality and magnitude of adaptive immune responses to vaccination. Lipid nanoparticle (LNP)-encapsulated nucleoside-modified mRNA vaccines have shown great efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanism of action of this vaccine platform is not well-characterized. Using influenza virus and SARS-CoV-2 mRNA and protein subunit vaccines, we demonstrated that our LNP formulation has intrinsic adjuvant activity that promotes induction of strong T follicular helper cell, germinal center B cell, long-lived plasma cell, and memory B cell responses that are associated with durable and protective antibodies in mice. Comparative experiments demonstrated that this LNP formulation outperformed a widely used MF59-like adjuvant, AddaVax. The adjuvant activity of the LNP relies on the ionizable lipid component and on IL-6 cytokine induction but not on MyD88- or MAVS-dependent sensing of LNPs. Our study identified LNPs as a versatile adjuvant that enhances the efficacy of traditional and next-generation vaccine platforms.

Germinal center responses to SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals.

Vaccine-mediated immunity often relies on the generation of protective antibodies and memory B cells, which commonly stem from germinal center (GC) reactions. An in-depth comparison of the GC responses elicited by SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals has not yet been performed due to the challenge of directly probing human lymph nodes. In this study, through a fine-needle-aspiration-based approach, we profiled the immune responses to SARS-CoV-2 mRNA vaccines in lymph nodes of healthy individuals and kidney transplant (KTX) recipients. We found that, unlike healthy subjects, KTX recipients presented deeply blunted SARS-CoV-2-specific GC B cell responses coupled with severely hindered T follicular helper cells, SARS-CoV-2 receptor-binding-domain-specific memory B cells and neutralizing antibodies. KTX recipients also displayed reduced SARS-CoV-2-specific CD4 and CD8 T cell frequencies. Broadly, these data indicate impaired GC-derived immunity in immunocompromised individuals, and suggest a GC-origin for certain humoral and memory B cell responses following mRNA vaccination.

SARS-CoV-2 mRNA Vaccines: Immunological Mechanism and Beyond.

To successfully protect against pathogen infection, a vaccine must elicit efficient adaptive immunity, including B and T cell responses. While B cell responses are key, as they can mediate antibody-dependent protection, T cells can modulate B cell activity and directly contribute to the elimination of pathogen-infected cells. In the unprecedented race to develop an effective vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the respiratory disease coronavirus disease 2019 (COVID-19), messenger RNA (mRNA) vaccines have emerged as front runners thanks to their capacity for rapid development and ability to drive potent adaptive immune responses. In this review article, we provide an overview of the results from pre-clinical studies in animal models as well as clinical studies in humans that assessed the efficacy of SARS-CoV-2 mRNA vaccines, with a primary focus on adaptive immune responses post vaccination.

CD4+ follicular regulatory T cells optimize the influenza virus-specific B cell response.

CD4+ follicular regulatory T (Tfr) cells control B cell responses through the modulation of follicular helper T (Tfh) cells and germinal center development while suppressing autoreactivity; however, their role in the regulation of productive germinal center B cell responses and humoral memory is incompletely defined. We show that Tfr cells promote antigen-specific germinal center B cell responses upon influenza virus infection. Following viral challenge, we found that Tfr cells are necessary for robust generation of virus-specific, long-lived plasma cells, antibody production against both hemagglutinin (HA) and neuraminidase (NA), the two major influenza virus glycoproteins, and appropriate regulation of the BCR repertoire. To further investigate the functional relevance of Tfr cells during viral challenge, we used a sequential immunization model with repeated exposure of antigenically partially conserved strains of influenza viruses, revealing that Tfr cells promote recall antibody responses against the conserved HA stalk region. Thus, Tfr cells promote antigen-specific B cell responses and are essential for the development of long-term humoral memory.

SARS-CoV-2 mRNA Vaccines Foster Potent Antigen-Specific Germinal Center Responses Associated with Neutralizing Antibody Generation.

The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms-mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant-looking for their abilities to quantitatively and qualitatively shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody production. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.

A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice.

SARS-CoV-2 infection has emerged as a serious global pandemic. Because of the high transmissibility of the virus and the high rate of morbidity and mortality associated with COVID-19, developing effective and safe vaccines is a top research priority. Here, we provide a detailed evaluation of the immunogenicity of lipid nanoparticle-encapsulated, nucleoside-modified mRNA (mRNA-LNP) vaccines encoding the full-length SARS-CoV-2 spike protein or the spike receptor binding domain in mice. We demonstrate that a single dose of these vaccines induces strong type 1 CD4+ and CD8+ T cell responses, as well as long-lived plasma and memory B cell responses. Additionally, we detect robust and sustained neutralizing antibody responses and the antibodies elicited by nucleoside-modified mRNA vaccines do not show antibody-dependent enhancement of infection in vitro. Our findings suggest that the nucleoside-modified mRNA-LNP vaccine platform can induce robust immune responses and is a promising candidate to combat COVID-19.

Harnessing Activin A Adjuvanticity to Promote Antibody Responses to BG505 HIV Envelope Trimers.

T follicular helper (TFH) cells are powerful regulators of affinity matured long-lived plasma cells. Eliciting protective, long-lasting antibody responses to achieve persistent immunity is the goal of most successful vaccines. Thus, there is potential in manipulating TFH cell responses. Herein, we describe an HIV vaccine development approach exploiting the cytokine activin A to improve antibody responses against recombinant HIV Envelope (Env) trimers in non-human primates. Administration of activin A improved the magnitude of Env-specific antibodies over time and promoted a significant increase in Env-specific plasma cells in the bone marrow. The boost in antibody responses was associated with reduced frequencies of T follicular regulatory (TFR) cells and increased germinal center T follicular helper (GC-TFH) to TFR cell ratios. Overall, these findings suggest that adjuvants inducing activin A production could potentially be incorporated in future rational design vaccine strategies aimed at improving germinal centers, long-lived plasma cells, and sustained antibody responses.

A distinct subpopulation of CD25- T-follicular regulatory cells localizes in the germinal centers.

T-follicular helper (Tfh) cells differentiate through a multistep process, culminating in germinal center (GC) localized GC-Tfh cells that provide support to GC-B cells. T-follicular regulatory (Tfr) cells have critical roles in the control of Tfh cells and GC formation. Although Tfh-cell differentiation is inhibited by IL-2, regulatory T (Treg) cell differentiation and survival depend on it. Here, we describe a CD25- subpopulation within both murine and human PD1+CXCR5+Foxp3+ Tfr cells. It is preferentially located in the GC and can be clearly differentiated from CD25+ non-GC-Tfr, Tfh, and effector Treg (eTreg) cells by the expression of a wide range of molecules. In comparison to CD25+ Tfr and eTreg cells, CD25- Tfr cells partially down-regulate IL-2-dependent canonical Treg features, but retain suppressive function, while simultaneously up-regulating genes associated with Tfh and GC-Tfh cells. We suggest that, similar to Tfh cells, Tfr cells follow a differentiation pathway generating a mature GC-localized subpopulation, CD25- Tfr cells.

Direct Probing of Germinal Center Responses Reveals Immunological Features and Bottlenecks for Neutralizing Antibody Responses to HIV Env Trimer.

Generating tier 2 HIV-neutralizing antibody (nAb) responses by immunization remains a challenging problem, and the immunological barriers to induction of such responses with Env immunogens remain unclear. Here, some rhesus monkeys developed autologous tier 2 nAbs upon HIV Env trimer immunization (SOSIP.v5.2) whereas others did not. This was not because HIV Env trimers were immunologically silent because all monkeys made similar ELISA-binding antibody responses; the key difference was nAb versus non-nAb responses. We explored the immunological barriers to HIV nAb responses by combining a suite of techniques, including longitudinal lymph node fine needle aspirates. Unexpectedly, nAb development best correlated with booster immunization GC B cell magnitude and Tfh characteristics of the Env-specific CD4 T cells. Notably, these factors distinguished between successful and unsuccessful antibody responses because GC B cell frequencies and stoichiometry to GC Tfh cells correlated with nAb development, but did not correlate with total Env Ab binding titers.