Co-formulation of the rF1V plague vaccine with depot-formulated cytokines enhances immunogenicity and efficacy to elicit protective responses against aerosol challenge in mice.

This study evaluated a depot-formulated cytokine-based adjuvant to improve the efficacy of the recombinant F1V (rF1V) plague vaccine and examined the protective response following aerosol challenge in a murine model. The results of this study showed that co-formulation of the Alhydrogel-adsorbed rF1V plague fusion vaccine with the depot-formulated cytokines recombinant human interleukin 2 (rhuIL-2) and/or recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF) significantly enhances immunogenicity and significant protection at lower antigen doses against a lethal aerosol challenge. These results provide additional support for the co-application of the depot-formulated IL-2 and/or GM-CSF cytokines to enhance vaccine efficacy.

OCA-B/Pou2af1 is sufficient to promote CD4+ T cell memory and prospectively identifies memory precursors.

The molecular mechanisms leading to the establishment of immunological memory are inadequately understood, limiting the development of effective vaccines and durable antitumor immune therapies. Here, we show that ectopic OCA-B expression is sufficient to improve antiviral memory recall responses, while having minimal effects on primary effector responses. At peak viral response, short-lived effector T cell populations are expanded but show increased Gadd45b and Socs2 expression, while memory precursor effector cells show increased expression of Bcl2, Il7r, and Tcf7 on a per-cell basis. Using an OCA-B mCherry reporter mouse line, we observe high OCA-B expression in CD4+ central memory T cells. We show that early in viral infection, endogenously elevated OCA-B expression prospectively identifies memory precursor cells with increased survival capability and memory recall potential. Cumulatively, the results demonstrate that OCA-B is both necessary and sufficient to promote CD4 T cell memory in vivo and can be used to prospectively identify memory precursor cells.

Effector-Phase IL-2 Signals Drive Th1 Effector and Memory Responses Dependently and Independently of TCF-1.

Following viral infection, CD4+ T cell differentiation is tightly regulated by cytokines and TCR signals. Although most activated CD4+ T cells express IL-2Rα after lymphocytic choriomeningtis virus infection, by day 3 postinfection, only half of activated T cells maintain expression. IL-2Rα at this time point distinguishes precursors for terminally differentiated Th1 cells (IL-2Rαhi) from precursors for Tfh cells and memory T cells (IL-2Rαlo) and is linked to strong TCR signals. In this study, we test whether TCR-dependent IL-2 links the TCR to CD4+ T cell differentiation. We employ a mixture of anti-IL-2 Abs to neutralize IL-2 throughout the primary CD4+ T cell response to lymphocytic choriomeningitis virus infection in mice or only after the establishment of lineage-committed effector cells (day 3 postinfection). We report that IL-2 signals drive the formation of Th1 precursor cells in the early stages of the immune response and sustain Th1 responses during its later stages (after day 3). Effector-stage IL-2 also shapes the composition and function of resulting CD4+ memory T cells. Although IL-2 has been shown previously to drive Th1 differentiation by reducing the activity of the transcriptional repressor TCF-1, we found that sustained IL-2 signals were still required to drive optimal Th1 differentiation even in the absence of TCF-1. Therefore, we concluded that IL-2 plays a central role throughout the effector phase in regulating the balance between Th1 and Tfh effector and memory cells via mechanisms that are both dependent and independent of its role in modulating TCF-1 activity.

Generation of antigen-specific memory CD4 T cells by heterologous immunization enhances the magnitude of the germinal center response upon influenza infection.

Current influenza vaccine strategies have yet to overcome significant obstacles, including rapid antigenic drift of seasonal influenza viruses, in generating efficacious long-term humoral immunity. Due to the necessity of germinal center formation in generating long-lived high affinity antibodies, the germinal center has increasingly become a target for the development of novel or improvement of less-efficacious vaccines. However, there remains a major gap in current influenza research to effectively target T follicular helper cells during vaccination to alter the germinal center reaction. In this study, we used a heterologous infection or immunization priming strategy to seed an antigen-specific memory CD4+ T cell pool prior to influenza infection in mice to evaluate the effect of recalled memory T follicular helper cells in increased help to influenza-specific primary B cells and enhanced generation of neutralizing antibodies. We found that heterologous priming with intranasal infection with acute lymphocytic choriomeningitis virus (LCMV) or intramuscular immunization with adjuvanted recombinant LCMV glycoprotein induced increased antigen-specific effector CD4+ T and B cellular responses following infection with a recombinant influenza strain that expresses LCMV glycoprotein. Heterologously primed mice had increased expansion of secondary Th1 and Tfh cell subsets, including increased CD4+ TRM cells in the lung. However, the early enhancement of the germinal center cellular response following influenza infection did not impact influenza-specific antibody generation or B cell repertoires compared to primary influenza infection. Overall, our study suggests that while heterologous infection/immunization priming of CD4+ T cells is able to enhance the early germinal center reaction, further studies to understand how to target the germinal center and CD4+ T cells specifically to increase long-lived antiviral humoral immunity are needed.

Tet2 deletion in CD4+ T cells disrupts Th1 lineage commitment in memory cells and enhances T follicular helper cell recall responses to viral rechallenge.

Following viral clearance, antigen-specific CD4+ T cells contract and form a pool of distinct Th1 and Tfh memory cells that possess unique epigenetic programs, allowing them to rapidly recall their specific effector functions upon rechallenge. DNA methylation programing mediated by the methylcytosine dioxygenase Tet2 contributes to balancing Th1 and Tfh cell differentiation during acute viral infection; however, the role of Tet2 in CD4+ T cell memory formation and recall is unclear. Using adoptive transfer models of antigen-specific wild type and Tet2 knockout CD4+ T cells, we find that Tet2 is required for full commitment of CD4+ T cells to the Th1 lineage and that in the absence of Tet2, memory cells preferentially recall a Tfh like phenotype with enhanced expansion upon secondary challenge. These findings demonstrate an important role for Tet2 in enforcing lineage commitment and programing proliferation potential, and highlight the potential of targeting epigenetic programing to enhance adaptive immune responses.

Immunogen-Specific Strengths and Limitations of the Activation-Induced Marker Assay for Assessing Murine Antigen-Specific CD4+ T Cell Responses.

The activation-induced marker (AIM) assay is a cytokine-independent technique to identify Ag-specific T cells based on the upregulated expression of activation markers after Ag restimulation. The method offers an alternative to intracellular cytokine staining in immunological studies, in which limited cytokine production makes the cell subsets of interest difficult to detect. Studies of lymphocytes in human and nonhuman primates have used the AIM assay to detect Ag-specific CD4+ and CD8+ T cells. However, there is a lack of validation of the strengths and limitations of the assay in murine (Mus musculus) models of infection and vaccination. In this study, we analyzed immune responses of TCR-transgenic CD4+ T cells, including lymphocytic choriomeningitis virus-specific SMARTA, OVA-specific OT-II, and diabetogenic BDC2.5-transgenic T cells, and measured the ability of the AIM assay to effectively identify these cells to upregulate AIM markers OX40 and CD25 following culture with cognate Ag. Our findings indicate that the AIM assay is effective for identifying the relative frequency of protein immunization-induced effector and memory CD4+ T cells, whereas the AIM assay had reduced ability to identify specific cells induced by viral infection, particularly during chronic lymphocytic choriomeningitis virus infection. Evaluation of polyclonal CD4+ T cell responses to acute viral infection demonstrated that the AIM assay can detect a proportion of both high- and low-affinity cells. Together, our findings indicate that the AIM assay can be an effective tool for relative quantification of murine Ag-specific CD4+ T cells to protein vaccination, while demonstrating its limitations during conditions of acute and chronic infection.

The magnitude of the germinal center B cell and T follicular helper cell response predicts long-lasting antibody titers to plague vaccination.

The development of a safe and effective vaccine against Yersinia pestis, the causative organism for plague disease, remains an important global health priority. Studies have demonstrated effective immune-based protection against plague challenge that is induced by plague antigen subunit vaccination in an aqueous alhydrogel formulation; however, whether these candidate vaccines in this formulation and presentation, induce long-lasting immunological memory in the form of durable cellular and antibody recall responses has not been fully demonstrated. In this study, we analyzed germinal center T follicular helper and germinal center B cell responses following F1V and F1 + V plague subunit immunization of mice with vaccines formulated in various adjuvants. Our data demonstrate that recombinant plague protein immunization formulated with IL-2/GM-CSF cytokines bound to alhydrogel adjuvant drive an increase in the magnitude of the germinal center T follicular helper and germinal center B cell responses following primary immunization, compared to vaccines formulated with Alhydrogel adjuvant alone. In contrast, plague protein subunit immunization combined with CpG ODN bound to alhydrogel increased the magnitude and duration of the germinal center Tfh and B cell responses following booster immunization. Importantly, enhanced germinal center Tfh and B cell responses correlated with long-lasting and high F1V-specific antibody titers and more robust antibody recall responses to F1V re-exposure. These findings indicate that vaccine formulations that drive enhancement of the germinal center Tfh and B cell responses are critical for inducing durable plague-specific humoral immunity.

Use of a MAIT-Activating Ligand, 5-OP-RU, as a Mucosal Adjuvant in a Murine Model of Vibrio cholerae O1 Vaccination.

Background: Mucosal-associated invariant T (MAIT) cells are innate-like T cells enriched in the mucosa with capacity for B-cell help. We hypothesize that targeting MAIT cells, using a MAIT-activating ligand as an adjuvant, could improve mucosal vaccine responses to bacterial pathogens such as Vibrio cholerae. Methods: We utilized murine models of V. cholerae vaccination to test the adjuvant potential of the MAIT-activating ligand, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU). We measured V. cholerae-specific antibody and antibody-secreting cell responses and used flow cytometry to examine MAIT-cell and B-cell phenotype, in blood, bronchoalveolar lavage fluid (BALF), and mucosal tissues, following intranasal vaccination with live V. cholerae O1 or a V. cholerae O1 polysaccharide conjugate vaccine. Results: We report significant expansion of MAIT cells in the lungs (P < 0.001) and BALF (P < 0.001) of 5-OP-RU treated mice, and higher mucosal (BALF, P = 0.045) but not systemic (serum, P = 0.21) V. cholerae O-specific-polysaccharide IgG responses in our conjugate vaccine model when adjuvanted with low-dose 5-OP-RU. In contrast, despite significant MAIT cell expansion, no significant differences in V. cholerae-specific humoral responses were found in our live V. cholerae vaccination model. Conclusions: Using a murine model, we demonstrate the potential, as well as the limitations, of targeting MAIT cells to improve antibody responses to mucosal cholera vaccines. Our study highlights the need for future research optimizing MAIT-cell targeting for improving mucosal vaccines.

Erratum to: Use of a MAIT-Activating Ligand, 5-OP-RU, as a Mucosal Adjuvant in a Murine Model of Vibrio cholerae O1 Vaccination.

[This corrects the article DOI: 10.20411/pai.v7i1.525.].

Tet2 coordinates with Foxo1 and Runx1 to balance T follicular helper cell and T helper 1 cell differentiation.

In response to various types of infection, naïve CD4+ T cells differentiate into diverse helper T cell subsets; however, the epigenetic programs that regulate differentiation in response to viral infection remain poorly understood. Demethylation of CpG dinucleotides by Tet methylcytosine dioxygenases is a key component of epigenetic programing that promotes specific gene expression, cellular differentiation, and function. We report that following viral infection, Tet2-deficient CD4+ T cells preferentially differentiate into highly functional germinal center T follicular helper (TFH) cells that provide enhanced help for B cells. Using genome-wide DNA methylation and transcription factor binding analyses, we find that Tet2 coordinates with multiple transcription factors, including Foxo1 and Runx1, to mediate the demethylation and expression of target genes, including genes encoding repressors of TFH differentiation. Our findings establish Tet2 as an important regulator of TFH cell differentiation and reveal pathways that could be targeted to enhance immune responses against infectious disease.

A subset of follicular helper-like MAIT cells can provide B cell help and support antibody production in the mucosa.

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that aid in protection against bacterial pathogens at mucosal surfaces through the release of inflammatory cytokines and cytotoxic molecules. Recent evidence suggests that MAIT cells can also provide B cell help. In this study, we describe a population of CXCR5+ T follicular helper (Tfh)­like MAIT cells (MAITfh) that have the capacity to provide B cell help within mucosal lymphoid organs. MAITfh cells are preferentially located near germinal centers in human tonsils and express the classical Tfh-associated transcription factor, B cell lymphoma 6 (BCL-6), the costimulatory markers inducible T cell costimulatory (ICOS) and programmed death receptor 1 (PD-1), and interleukin-21 (IL-21). We demonstrate the ability of MAIT cells to provide B cell help in vivo after mucosal challenge with Vibrio cholerae. Specifically, we show that adoptive transfer of MAIT cells into αß T cell­deficient mice promoted B cell differentiation and increased serum V. cholerae­specific IgA responses. Our data demonstrate the capacity of MAIT cells to participate in adaptive immune responses and suggest that MAIT cells may be potential targets for mucosal vaccines.

Safety assessment of Streptococcus salivarius M18 a probiotic for oral health.

The development of probiotics targeting non-intestinal body sites continues to generate interest amongst researchers, biotech companies and consumers alike. A key consideration for any bacterial strain to be developed into a probiotic is a robust assessment of its safety profile. Streptococcus salivarius strain M18 was originally isolated from a healthy adult and evaluated for its probiotic capabilities targeted to dental and oral health applications. This publication presents the safety characterisation of strain M18. Application of a diverse range of techniques showed that strain M18 can be specifically distinguished from other S. salivarius using a variety of molecular and phenotypic methodologies and that it lacks any relevant antibiotic resistance or virulence determinants. Direct comparison of the strain M18 safety profile with that of the prototype S. salivarius probiotic, S. salivarius strain K12, supports the proposition that strain M18 is indeed safe for probiotic application in humans.

Protein Immunization Induces Memory CD4+ T Cells That Lack Th Lineage Commitment.

Acute viral infection generates lineage-committed Th1 and T follicular helper (Tfh) memory cells that recall their lineage-specific functions following secondary challenge with virus. However, the lineage commitment of effector and memory Th cells in vivo following protein vaccination is poorly understood. In this study, we analyzed effector and memory CD4+ T cell differentiation in mice (Mus musculus) following adjuvanted glycoprotein immunization compared with acute lymphocytic choriomeningitis virus infection. Glycoprotein immunization induced CXCR5- non-Tfh effector and memory CD4+ T cells that surprisingly had not undergone polarization toward any particular Th cell lineage but had undergone memory differentiation. However, upon challenge with virus, these Th lineage-nonpolarized memory CD4+ T cells were able to generate Th1 secondary effector cells, demonstrating their lineage plasticity. In addition, Tfh and memory Tfh cells were generated in response to protein immunization, and these cells differed from infection-induced Tfh cells by their lack of the transcription factor Tbet. Rechallenge experiments demonstrated that viral infection, but not protein immunization, during either the primary or secondary immune response, restricts the recall of Bcl6 expression and the generation of germinal center Tfh cells. Together, these data demonstrate that protein immunization generates a combination of nonpolarized memory cells that are highly plastic and memory Tfh cells that can undergo further Th1-like modulation during a secondary response to viral infection.

Cytokine-skewed Tfh cells: functional consequences for B cell help.

CD4+ follicular helper T (Tfh) cells play a vital role in providing help for B cells undergoing selection and differentiation into activated antibody-secreting cells in mammalian germinal centers (GCs). Increasing evidence suggests that Tfh cells are a heterogeneous population that generates cytokine-skewed immune responses - a reflection of the microenvironment during differentiation. This has important ramifications for Tfh-mediated B cell help. Because Tfh subsets can have opposing effects on GC B cell responses, we discuss current findings regarding the differentiation and functions of cytokine-skewed Tfh cells in modulating GC B cell differentiation. Antibodies are important weapons against infectious diseases but can also be pathogenic mediators in some autoimmune conditions. Since cytokine-skewed Tfh cells can influence the magnitude and quality of the humoral response, we address the roles of cytokine-skewed Tfh cells in disease.

Mucosal-associated invariant T (MAIT) cells mediate protective host responses in sepsis.

Sepsis is a systemic inflammatory response to infection and a leading cause of death. Mucosal-associated invariant T (MAIT) cells are innate-like T cells enriched in mucosal tissues that recognize bacterial ligands. We investigated MAIT cells during clinical and experimental sepsis, and their contribution to host responses. In experimental sepsis, MAIT-deficient mice had significantly increased mortality and bacterial load, and reduced tissue-specific cytokine responses. MAIT cells of WT mice expressed lower levels of IFN-γ and IL-17a during sepsis compared to sham surgery, changes not seen in non-MAIT T cells. MAIT cells of patients at sepsis presentation were significantly reduced in frequency compared to healthy donors, and were more activated, with decreased IFN-γ production, compared to both healthy donors and paired 90-day samples. Our data suggest that MAIT cells are highly activated and become dysfunctional during clinical sepsis, and contribute to tissue-specific cytokine responses that are protective against mortality during experimental sepsis.

Joint range of motion entropy changes in response to load carriage in military personnel.

BACKGROUND: Overuse accounts for 82% of injuries in military personnel, and these occur predominantly in the spine and lower limbs. While non-linear analyses have shown changes in overall stability of the movement during load carriage, individual joint contributions have not been studied. The concept of entropy compensation between task, organism and environmental constraints is studied at a joint level. RESEARCH QUESTION: The aim of this study was to investigate whether using different methods of loading by military personnel would have an effect on the sample entropy of the joint ranges of motion. METHODS: Eleven male reserve infantry army soldiers (age: 22 ±â€¯2 years; height: 1.80 ±â€¯0.06 m; mass: 89.3 ±â€¯14.4 kg) walked an outdoor, 800 m course under 5 load conditions: unloaded, 15 kg backpack, 25 kg backpack, 15 kg webbing and backpack and 25 kg webbing and backpack. Kinematic data was recorded at 240 Hz using the Xsens motion capture system. The ranges of motion (ROM) of the spine, hips and knee were calculated for each gait cycle. Mean ROM, coefficient of variation (CV) of the ROM and the sample entropy of the ROM were compared between conditions. RESULTS: Spine side flexion ROM decreased significantly from the control condition in all loaded conditions, while sample entropy of the spine side flexion ROM increased in some conditions with no significant change in CV. Conversely, the hip flexion ROM increased significantly from the control, while sample entropy of the hip flexion ROM decreased. SIGNIFICANCE: These results suggest that entropy compensation may propagate at a joint level. Understanding that a decrease in certainty with which a joint angle is selected, may be accompanied by an increase at a neighbouring joint. This could be significant in monitoring injuries as a result of environmental or task constraints.

Recurrent Tonsillitis Tfh Cells Acquire a Killer Identity.

Group A Streptococcus (GAS) infection causes recurrent tonsillitis (RT) in some individuals. A recent study (Dan et al. Sci. Transl. Med. 2019;11:eaau3776) demonstrates that RT is associated with an impaired antibody response against a key streptococcal virulence factor. This factor, SpeA, can induce abnormal T follicular helper (Tfh) cells that are able to kill B cells.

Effector CD8 T cells dedifferentiate into long-lived memory cells.

Memory CD8 T cells that circulate in the blood and are present in lymphoid organs are an essential component of long-lived T cell immunity. These memory CD8 T cells remain poised to rapidly elaborate effector functions upon re-exposure to pathogens, but also have many properties in common with naive cells, including pluripotency and the ability to migrate to the lymph nodes and spleen. Thus, memory cells embody features of both naive and effector cells, fuelling a long-standing debate centred on whether memory T cells develop from effector cells or directly from naive cells. Here we show that long-lived memory CD8 T cells are derived from a subset of effector T cells through a process of dedifferentiation. To assess the developmental origin of memory CD8 T cells, we investigated changes in DNA methylation programming at naive and effector cell-associated genes in virus-specific CD8 T cells during acute lymphocytic choriomeningitis virus infection in mice. Methylation profiling of terminal effector versus memory-precursor CD8 T cell subsets showed that, rather than retaining a naive epigenetic state, the subset of cells that gives rise to memory cells acquired de novo DNA methylation programs at naive-associated genes and became demethylated at the loci of classically defined effector molecules. Conditional deletion of the de novo methyltransferase Dnmt3a at an early stage of effector differentiation resulted in reduced methylation and faster re-expression of naive-associated genes, thereby accelerating the development of memory cells. Longitudinal phenotypic and epigenetic characterization of the memory-precursor effector subset of virus-specific CD8 T cells transferred into antigen-free mice revealed that differentiation to memory cells was coupled to erasure of de novo methylation programs and re-expression of naive-associated genes. Thus, epigenetic repression of naive-associated genes in effector CD8 T cells can be reversed in cells that develop into long-lived memory CD8 T cells while key effector genes remain demethylated, demonstrating that memory T cells arise from a subset of fate-permissive effector T cells.

Human mucosal-associated invariant T (MAIT) cells possess capacity for B cell help.

Mucosal-associated invariant T (MAIT) cells are an innate-like T cell subset, restricted by the nonclassic MHC class I-related protein MR1 and enriched at mucosal sites. Human studies have shown an association between MAIT cells and pathogen-specific antibody responses. In this study, we investigate the effect of human MAIT cells on B cells ex vivo. We found that supernatants from microbe- or cytokine-stimulated MAIT cells, when added to purified autologous B cells, increase frequencies of plasmablasts and promote IgA, IgG, and IgM production. We found effects to be mostly MR1-dependent and that the increases in plasmablasts are likely a result of increased differentiation from memory B cells. Furthermore, microbe-activated MAIT cell supernatant contains multiple cytokines known to stimulate B cells, including IL-6, -10, and -21. This study thus provides the first direct evidence of a newly identified role of MAIT cells in providing help to B cells.

Antigen Density Dictates Immune Responsiveness following Red Blood Cell Transfusion.

Although RBC transfusion can result in the development of anti-RBC alloantibodies that increase the probability of life-threatening hemolytic transfusion reactions, not all patients generate anti-RBC alloantibodies. However, the factors that regulate immune responsiveness to RBC transfusion remain incompletely understood. One variable that may influence alloantibody formation is RBC alloantigen density. RBC alloantigens exist at different densities on the RBC surface and likewise exhibit distinct propensities to induce RBC alloantibody formation. However, although distinct alloantigens reside on the RBC surface at different levels, most alloantigens also represent completely different structures, making it difficult to separate the potential impact of differences in Ag density from other alloantigen features that may also influence RBC alloimmunization. To address this, we generated RBCs that stably express the same Ag at different levels. Although exposure to RBCs with higher Ag levels induces a robust Ab response, RBCs bearing low Ag levels fail to induce RBC alloantibodies. However, exposure to low Ag-density RBCs is not without consequence, because recipients subsequently develop Ag-specific tolerance. Low Ag-density RBC-induced tolerance protects higher Ag-density RBCs from immune-mediated clearance, is Ag specific, and occurs through the induction of B cell unresponsiveness. These results demonstrate that Ag density can potently impact immune outcomes following RBC transfusion and suggest that RBCs with altered Ag levels may provide a unique tool to induce Ag-specific tolerance.