Publications by authors named "Andrea J Sant"

70 Publications

Circulating CD4 T cells elicited by endemic coronaviruses display vast disparities in abundance and functional potential linked to both antigen specificity and age.

J Infect Dis 2021 Feb 8. Epub 2021 Feb 8.

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, United States of America.

Repeated infections with endemic human coronaviruses are thought to reflect lack of long-lasting protective immunity. Here, we evaluate circulating human CD4 T cells collected prior to 2020 for reactivity towards hCoV spike proteins, probing for the ability to produce IFN-γ, IL-2 or granzyme B. We find robust reactivity to spike-derived epitopes, comparable to influenza, but highly variable abundance and functional potential across subjects, depending on age and viral antigen specificity. To explore the potential of these memory cells to be recruited in SARS-CoV-2 infection, we examined the same subjects for cross-reactive recognition of epitopes from SARS-CoV-2 nucleocapsid, membrane/envelope, and spike. The functional potential of these cross-reactive CD4 T cells was highly variable, with nucleocapsid-specific CD4 T cells, but not spike-reactive cells showing exceptionally high levels of granzyme production upon stimulation. These results are considered in light of recruitment of hCoV-reactive cells into responses of humans to SARS-CoV infections or vaccinations.
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http://dx.doi.org/10.1093/infdis/jiab076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928818PMC
February 2021

Heterologous viral protein interactions within licensed seasonal influenza virus vaccines.

NPJ Vaccines 2020 Jan 10;5(1). Epub 2020 Jan 10.

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA.

Currently, licensed influenza virus vaccines are designed and tested only for their ability to elicit hemagglutinin (HA)-reactive, neutralizing antibodies. Despite this, the purification process in vaccine manufacturing often does not completely remove other virion components. In the studies reported here, we have examined the viral protein composition of a panel of licensed vaccines from different manufacturers and licensed in different years. Using western blotting, we found that, beyond HA proteins, there are detectable quantities of neuraminidase (NA), nucleoprotein (NP), and matrix proteins (M1) from both influenza A and influenza B viruses in the vaccines but that the composition differed by source and method of vaccine preparation. We also found that disparities in viral protein composition were associated with distinct patterns of elicited antibody specificities. Strikingly, our studies also revealed that many viral proteins contained in the vaccine form heterologous complexes. When H1 proteins were isolated by immunoprecipitation, NA (N1), M1 (M1-A), H3, and HA-B proteins were co-isolated with the H1. Further biochemical studies suggest that these interactions persist for at least 4 h at 37 °C and that the membrane/intracytoplasmic domains in the intact HA proteins are important for the intermolecular interactions detected. These studies indicate that, if such interactions persist after vaccines reach the draining lymph node, both dendritic cells and HA-specific B cells may take up multiple viral proteins simultaneously. Whether these interactions are beneficial or harmful to the developing immune response will depend on the functional potential of the elicited virus-specific CD4 T cells.
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http://dx.doi.org/10.1038/s41541-019-0153-1DOI Listing
January 2020

Recombinant HA-based vaccine outperforms split and subunit vaccines in elicitation of influenza-specific CD4 T cells and CD4 T cell-dependent antibody responses in humans.

NPJ Vaccines 2020 26;5:77. Epub 2020 Aug 26.

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Ave, Box 609, Rochester, NY 14642 USA.

Although traditional egg-based inactivated influenza vaccines can protect against infection, there have been significant efforts to develop improved formats to overcome disadvantages of this platform. Here, we have assessed human CD4 T cell responses to a traditional egg-based influenza vaccine with recently available cell-derived vaccines and recombinant baculovirus-derived vaccines. Adults were administered either egg-derived Fluzone, mammalian cell-derived Flucelvax or recombinant HA (Flublok). CD4 T cell responses to each HA protein were assessed by cytokine EliSpot and intracellular staining assays. The specificity and magnitude of antibody responses were quantified by ELISA and HAI assays. By all criteria, Flublok vaccine exhibited superior performance in eliciting both CD4 T cell responses and HA-specific antibody responses, whether measured by mean response magnitude or percent of responders. Although the mechanism(s) underlying this advantage is not yet clear, it is likely that both qualitative and quantitative features of the vaccines impact the response.
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http://dx.doi.org/10.1038/s41541-020-00227-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450042PMC
August 2020

Heterologous viral protein interactions within licensed seasonal influenza virus vaccines.

NPJ Vaccines 2020 10;5. Epub 2020 Jan 10.

1David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY USA.

Currently, licensed influenza virus vaccines are designed and tested only for their ability to elicit hemagglutinin (HA)-reactive, neutralizing antibodies. Despite this, the purification process in vaccine manufacturing often does not completely remove other virion components. In the studies reported here, we have examined the viral protein composition of a panel of licensed vaccines from different manufacturers and licensed in different years. Using western blotting, we found that, beyond HA proteins, there are detectable quantities of neuraminidase (NA), nucleoprotein (NP), and matrix proteins (M1) from both influenza A and influenza B viruses in the vaccines but that the composition differed by source and method of vaccine preparation. We also found that disparities in viral protein composition were associated with distinct patterns of elicited antibody specificities. Strikingly, our studies also revealed that many viral proteins contained in the vaccine form heterologous complexes. When H1 proteins were isolated by immunoprecipitation, NA (N1), M1 (M1-A), H3, and HA-B proteins were co-isolated with the H1. Further biochemical studies suggest that these interactions persist for at least 4 h at 37 °C and that the membrane/intracytoplasmic domains in the intact HA proteins are important for the intermolecular interactions detected. These studies indicate that, if such interactions persist after vaccines reach the draining lymph node, both dendritic cells and HA-specific B cells may take up multiple viral proteins simultaneously. Whether these interactions are beneficial or harmful to the developing immune response will depend on the functional potential of the elicited virus-specific CD4 T cells.
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http://dx.doi.org/10.1038/s41541-019-0153-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954117PMC
January 2020

Peptide Epitope Hot Spots of CD4 T Cell Recognition Within Influenza Hemagglutinin During the Primary Response to Infection.

Pathogens 2019 Nov 5;8(4). Epub 2019 Nov 5.

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.

Antibodies specific for the hemagglutinin (HA) protein of influenza virus are critical for protective immunity to infection. Our studies show that CD4 T cells specific for epitopes derived from HA are the most effective in providing help for the HA-specific B cell responses to infection and vaccination. In this study, we asked whether HA epitopes recognized by CD4 T cells in the primary response to infection are equally distributed across the HA protein or if certain segments are enriched in CD4 T cell epitopes. Mice that collectively expressed eight alternative MHC (Major Histocompatibility Complex) class II molecules, that would each have different peptide binding specificities, were infected with an H1N1 influenza virus. CD4 T cell peptide epitope specificities were identified by cytokine EliSpots. These studies revealed that the HA-specific CD4 T cell epitopes cluster in two distinct regions of HA and that some segments of HA are completely devoid of CD4 T cell epitopes. When located on the HA structure, it appears that the regions that most poorly recruit CD4 T cells are sequestered within the interior of the HA trimer, perhaps inaccessible to the proteolytic machinery inside the endosomal compartments of antigen presenting cells.
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http://dx.doi.org/10.3390/pathogens8040220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963931PMC
November 2019

Evidence That Blunted CD4 T-Cell Responses Underlie Deficient Protective Antibody Responses to Influenza Vaccines in Repeatedly Vaccinated Human Subjects.

J Infect Dis 2020 06;222(2):273-277

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, New York.

Despite the benefits of yearly influenza vaccination, accumulating evidence suggests that diminished vaccine efficacy may be related to repeated vaccination. Although studied at the level of B-cell responses, CD4 T-cell responses have not yet been examined. In this study, we analyze CD4 T-cell responses to influenza vaccination in subjects who differ in their vaccine history. We find a striking disparity in their responses, with previously vaccinated subjects exhibiting significantly blunted CD4 T-cell responses and diminished antibody responses. These results suggest that limiting CD4 T-cell help mteaserrlie the diminished or altered antibody responses in repeatedly vaccinated subjects.
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http://dx.doi.org/10.1093/infdis/jiz433DOI Listing
June 2020

Monoclonal Antibody Responses after Recombinant Hemagglutinin Vaccine versus Subunit Inactivated Influenza Virus Vaccine: a Comparative Study.

J Virol 2019 11 15;93(21). Epub 2019 Oct 15.

Department of Medicine, Section of Rheumatology, The University of Chicago, Chicago, Illinois, USA

Vaccination is the best measure of protection against influenza virus infection. Vaccine-induced antibody responses target mainly the hemagglutinin (HA) surface glycoprotein, composed of the head and the stalk domains. Recently two novel vaccine platforms have been developed for seasonal influenza vaccination: a recombinant HA vaccine produced in insect cells (Flublok) and Flucelvax, prepared from virions produced in mammalian cells. In order to compare the fine specificity of the antibodies induced by these two novel vaccine platforms, we characterized 42 Flublok-induced monoclonal antibodies (MAbs) and 38 Flucelvax-induced MAbs for avidity, cross-reactivity, and any selectivity toward the head versus the stalk domain. These studies revealed that Flublok induced a greater proportion of MAbs targeting epitopes near the receptor-binding domain on HA head (hemagglutinin inhibition-positive MAbs) than Flucelvax, while the two vaccines induced similar low frequencies of stalk-reactive MAbs. Finally, mice immunized with Flublok and Flucelvax also induced similar frequencies of stalk-reactive antibody-secreting cells, showing that HA head immunodominance is independent of immune memory bias. Collectively, our results suggest that these vaccine formulations are similarly immunogenic but differ in the preferences of the elicited antibodies toward the receptor-binding domain on the HA head. There are ongoing efforts to increase the efficacy of influenza vaccines and to promote production strategies that can rapidly respond to newly emerging viruses. It is important to understand if current alternative seasonal vaccines, such as Flublok and Flucelvax, that use alternate production strategies can induce protective influenza-specific antibodies and to evaluate what type of epitopes are targeted by distinct vaccine formulations.
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http://dx.doi.org/10.1128/JVI.01150-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803255PMC
November 2019

A Novel Vaccine Strategy to Overcome Poor Immunogenicity of Avian Influenza Vaccines through Mobilization of Memory CD4 T Cells Established by Seasonal Influenza.

J Immunol 2019 09 9;203(6):1502-1508. Epub 2019 Aug 9.

Department of Microbiology and Immunology, D.H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY 14642; and

Avian influenza vaccines exhibit poor immunogenicity in humans. We hypothesized that one factor underlying weak B cell responses was sequence divergence between avian and seasonal influenza hemagglutinin proteins, thus limiting the availability of adequate CD4 T cell help. To test this, a novel chimeric hemagglutinin protein (cH7/3) was derived, comprised of the stem domain from seasonal H3 hemagglutinin and the head domain from avian H7. Immunological memory to seasonal influenza was established in mice, through strategies that included seasonal inactivated vaccines, Flumist, and synthetic peptides derived from the H3 stalk domain. After establishment of memory, mice were vaccinated with H7 or cH7/3 protein. The cH7/3 Ag was able to recall H3-specific CD4 T cells, and this potentiated CD4 T cell response was associated with enhanced early germinal center response and rapid elicitation of Abs to H7, including Abs specific for the H7 head domain. These results suggest that in pandemic situations, inclusion of CD4 T cell epitopes from seasonal viruses have the potential to overcome the poor immunogenicity of avian vaccines by helping B cells and conferring greater subtype-specific Ab response to viral HA.
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http://dx.doi.org/10.4049/jimmunol.1900819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737897PMC
September 2019

Protein Vaccination Directs the CD4 T Cell Response toward Shared Protective Epitopes That Can Be Recalled after Influenza Virus Infection.

J Virol 2019 10 30;93(20). Epub 2019 Sep 30.

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA

Vaccination is widely used to generate protective immunity against influenza virus. CD4 T cells contribute in diverse ways to protective immunity, most notably, in the provision of help for the production of neutralizing antibodies. Several recent reports have suggested that influenza virus infection elicits CD4 T cells whose specificity only partially overlaps that of T cells elicited by vaccination. This finding has raised serious concerns regarding the utility of currently licensed inactivated influenza virus vaccines and novel protein-based vaccines. Here, using controlled animal models that allowed a broad sampling of the CD4 T cell repertoire, we evaluated protein vaccine- versus infection-generated CD4 T cell epitopes. Our studies revealed that all the infection-elicited CD4 T cell epitope specificities are also elicited by protein vaccination, although the immunodominance hierarchies can differ. Finally, using a reverse-engineered influenza virus and a heterologous protein vaccination and infection challenge strategy, we show that protein vaccine-elicited CD4 memory T cells are recalled and boosted after infection and provide early help to accelerate hemagglutinin (HA)-specific antibody responses. The early CD4 T cell response and HA-specific antibody production are associated with lowered viral titers during the infection challenge. Our data lend confidence to the ability of current protein-based vaccines to elicit influenza virus-specific CD4 T cells that can potentiate protective immunity upon influenza virus infection. Most current and new influenza vaccine candidates consist of a single influenza virus protein or combinations of influenza virus proteins. For these vaccines to elicit CD4 T cells that can be recalled after infection, the peptide epitopes should be shared between the two modes of confrontation. Recently, questions regarding the relatedness of epitope selection by influenza virus infection and protein vaccination have been raised. However, the studies reported here show that the specificity of CD4 T cells elicited by protein-based vaccines overlaps that of T cells elicited by infection and that CD4 T cells primed by protein vaccines are recalled and contribute to protection of the host from a future infection.
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http://dx.doi.org/10.1128/JVI.00947-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798096PMC
October 2019

Imprinting and Editing of the Human CD4 T Cell Response to Influenza Virus.

Front Immunol 2019 7;10:932. Epub 2019 May 7.

Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY, United States.

Immunity to influenza is unique among pathogens, in that immune memory is established both via intermittent lung localized infections with highly variable influenza virus strains and by intramuscular vaccinations with inactivated protein-based vaccines. Studies in the past decades have suggested that the B cell responses to influenza infection and vaccination are highly biased by an individual's early history of influenza infection. This reactivity likely reflects both the competitive advantage that memory B cells have in an immune response and the relatively limited diversity of epitopes in influenza hemagglutinin that are recognized by B cells. In contrast, CD4 T cells recognize a wide array of epitopes, with specificities that are heavily influenced by the diversity of influenza antigens available, and a multiplicity of functions that are determined by both priming events and subsequent confrontations with antigens. Here, we consider the events that prime and remodel the influenza-specific CD4 T cell response in humans that have highly diverse immune histories and how the CD4 repertoire may be edited in terms of functional potential and viral epitope specificity. We discuss the consequences that imprinting and remodeling may have on the potential of different human hosts to rapidly respond with protective cellular immunity to infection. Finally, these issues are discussed in the context of future avenues of investigation and vaccine strategies.
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http://dx.doi.org/10.3389/fimmu.2019.00932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514101PMC
September 2020

Editorial: Orchestration of an Immune Response to Respiratory Pathogens.

Front Immunol 2019 9;10:690. Epub 2019 Apr 9.

Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, Rochester, NY, United States.

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http://dx.doi.org/10.3389/fimmu.2019.00690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465544PMC
August 2020

The Way Forward: Potentiating Protective Immunity to Novel and Pandemic Influenza Through Engagement of Memory CD4 T Cells.

Authors:
Andrea J Sant

J Infect Dis 2019 04;219(Suppl_1):S30-S37

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, New York.

Potentially pandemic strains of influenza pose an undeniable threat to human populations. Therefore, it is essential to develop better strategies to enhance vaccine design and predict parameters that identify susceptible humans. CD4 T cells are a central component of protective immunity to influenza, delivering direct effector function and potentiating responses of other lymphoid cells. Humans have highly diverse influenza-specific CD4 T-cell populations that vary in stimulation history, specificity, and functionality. These complexities constitute a formidable obstacle to predicting immune responses to pandemic strains of influenza and derivation of optimal vaccine strategies. We suggest that more precise efforts to identify and enumerate both the positive and negative contributors of immunity in the CD4 T-cell compartment will aid in both predicting susceptible hosts and in development of vaccination strategies that will poise most human subjects to respond to pandemic influenza strains with protective immune responses.
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http://dx.doi.org/10.1093/infdis/jiy666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452298PMC
April 2019

Analyses of Cellular Immune Responses in Ferrets Following Influenza Virus Infection.

Methods Mol Biol 2018 ;1836:513-530

Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY, USA.

Ferrets are an ideal animal model in which to study the transmission of respiratory viruses as well as disease progression and vaccine efficacy because of their close anatomical and physiological resemblances to humans. However, a paucity of reagents and standardized procedures has hampered research progress, especially for studying cell-mediated immunity. The approaches described here-leukocyte isolation from whole blood and secondary lymphoid tissues-are generalizable, highly reproducible, and deliver single cell suspensions with excellent cell viability. Importantly, we have now developed assays to quantify key cellular components and antigen-specific T cell responses at the single cell level from multiple tissue compartments following influenza infection in ferrets. Collectively, these methods were instrumental in flow cytometry studies that revealed alterations in immune cell composition and distribution across lymphoid tissues following viral infection. Furthermore, sorting of T cell populations and peptide restimulation ex vivo in cytokine ELISpot assays has provided novel insight into the influenza-specific CD4 and CD8 T cell repertoire. The detailed procedures for these techniques are described in this chapter and can likely be adapted for the analyses of responses to many respiratory pathogens.
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http://dx.doi.org/10.1007/978-1-4939-8678-1_24DOI Listing
April 2019

Moving Forward: Recent Developments for the Ferret Biomedical Research Model.

mBio 2018 07 17;9(4). Epub 2018 Jul 17.

Department of Immunology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

Since the initial report in 1911, the domestic ferret has become an invaluable biomedical research model. While widely recognized for its utility in influenza virus research, ferrets are used for a variety of infectious and noninfectious disease models due to the anatomical, metabolic, and physiological features they share with humans and their susceptibility to many human pathogens. However, there are limitations to the model that must be overcome for maximal utility for the scientific community. Here, we describe important recent advances that will accelerate biomedical research with this animal model.
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http://dx.doi.org/10.1128/mBio.01113-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6050969PMC
July 2018

CD4 T cells in protection from influenza virus: Viral antigen specificity and functional potential.

Immunol Rev 2018 07;284(1):91-105

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA.

CD4 T cells convey a number of discrete functions to protective immunity to influenza, a complexity that distinguishes this arm of adaptive immunity from B cells and CD8 T cells. Although the most well recognized function of CD4 T cells is provision of help for antibody production, CD4 T cells are important in many aspects of protective immunity. Our studies have revealed that viral antigen specificity is a key determinant of CD4 T cell function, as illustrated both by mouse models of infection and human vaccine responses, a factor whose importance is due at least in part to events in viral antigen handling. We discuss research that has provided insight into the diverse viral epitope specificity of CD4 T cells elicited after infection, how this primary response is modified as CD4 T cells home to the lung, establish memory, and after challenge with a secondary and distinct influenza virus strain. Our studies in human subjects point out the challenges facing vaccine efforts to facilitate responses to novel and avian strains of influenza, as well as strategies that enhance the ability of CD4 T cells to promote protective antibody responses to both seasonal and potentially pandemic strains of influenza.
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http://dx.doi.org/10.1111/imr.12662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6070306PMC
July 2018

Overarching Immunodominance Patterns and Substantial Diversity in Specificity and Functionality in the Circulating Human Influenza A and B Virus-Specific CD4+ T-Cell Repertoire.

J Infect Dis 2018 08;218(7):1169-1174

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, New York.

There is limited information on the antigen specificity and functional potential of the influenza virus-specific CD4+ T-cell repertoire in humans. Here, enzyme-linked immunospot assays were used to examine circulating CD4+ T-cell specificities for influenza virus directly ex vivo in healthy adults. Our studies revealed CD4+ T-cell reactivity to multiple influenza virus proteins, including hemagglutinins, neuraminidases, M1 proteins, and nucleoproteins. Unexpectedly, the immunodominance hierarchies and functional potential of cells reactive toward influenza A virus were distinct from those toward influenza B virus. We also identified influenza virus-specific cells producing granzyme B. Our findings revealed individual and virus-specific patterns that may differentially poise humans to respond to infection or vaccination.
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http://dx.doi.org/10.1093/infdis/jiy288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6455892PMC
August 2018

Diverse Epitope Specificity, Immunodominance Hierarchy, and Functional Avidity of Effector CD4 T Cells Established During Priming Is Maintained in Lung After Influenza A Virus Infection.

Front Immunol 2018 6;9:655. Epub 2018 Apr 6.

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, United States.

One of the major contributions to protective immunity to influenza viruses that is provided by virus-specific CD4 T cells is delivery of effector function to the infected lung. However, there is little known about the selection and breadth of viral epitope-specific CD4 T cells that home to the lung after their initial priming. In this study, using a mouse model of influenza A infection and an unbiased method of epitope identification, the viral epitope-specific CD4 T cells elicited after infection were identified and quantified. We found that a very diverse specificity of CD4 T cells is primed by infection, including epitopes from hemagglutinin, neuraminidase, matrix protein, nucleoprotein, and non-structural protein-1. Using peptide-specific cytokine EliSpots, the diversity and immunodominance hierarchies established in the lung-draining lymph node were compared with specificities of CD4 T cells that home to the lung. Our studies revealed that CD4 T cells of all epitope specificities identified in peripheral lymphoid tissue home back to the lung and that most of these lung-homing cells are localized within the tissue rather than the pulmonary vasculature. There is a striking shift of CD4 T cell functionality that enriches for IFN-γ production as cells are primed in the lymph node, enter the lung vasculature, and finally establish residency in the tissue, but with no apparent shifts in their functional avidity. We conclude that CD4 T cells of broad viral epitope specificity are recruited into the lung after influenza infection, where they then have the opportunity to encounter infected or antigen-bearing antigen-presenting cells.
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http://dx.doi.org/10.3389/fimmu.2018.00655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897437PMC
June 2019

CD4 T Cell Epitope Specificity and Cytokine Potential Are Preserved as Cells Transition from the Lung Vasculature to Lung Tissue following Influenza Virus Infection.

J Virol 2018 07 13;92(13). Epub 2018 Jun 13.

Department of Microbiology and Immunology, D. H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, New York, USA

Pulmonary CD4 T cells are critical in respiratory virus control, both by delivering direct effector function and through coordinating responses of other immune cells. Recent studies have shown that following influenza virus infection, virus-specific CD4 T cells are partitioned between pulmonary vasculature and lung tissue. However, very little is known about the peptide specificity or functional differences of CD4 T cells within these two compartments. Using a mouse model of influenza virus infection in conjunction with intravascular labeling , the cell surface phenotype, epitope specificity, and functional potential of the endogenous polyclonal CD4 T cell response was examined by tracking nine independent CD4 T cell epitope specificities. These studies revealed that tissue-localized CD4 cells were globally distinct from vascular cells in expression of markers associated with transendothelial migration, residency, and micropositioning. Despite these differences, there was little evidence for remodeling of the viral epitope specificity or cytokine potential as cells transition from vasculature to the highly inflamed lung tissue. Our studies also distinguished cells in the pulmonary vasculature from peripheral circulating CD4 T cells, providing support for the concept that the pulmonary vasculature does not simply reflect circulating cells that are trapped within the narrow confines of capillary vessels but rather is enriched in transitional cells primed in the draining lymph node that have specialized potential to enter the lung tissue. CD4 T cells convey a multitude of functions in immunity to influenza, including those delivered in the lymph node and others conveyed by CD4 T cells that leave the lymph node, enter the blood, and extravasate into the lung tissue. Here, we show that the transition of recently primed CD4 cells detected in the lung vasculature undergo profound changes in expression of markers associated with tissue localization as they establish residence in the lung. However, this transition does not edit CD4 T cell epitope specificity or the cytokine potential of the CD4 T cells. Thus, CD4 T cells that enter the infected lung can convey diverse functions and have a sufficiently broad viral antigen specificity to detect the complex array of infected cells within the infected tissue, offering the potential for more effective protective function.
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http://dx.doi.org/10.1128/JVI.00377-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002724PMC
July 2018

Distinct and complementary roles of CD4 T cells in protective immunity to influenza virus.

Curr Opin Immunol 2018 08 2;53:13-21. Epub 2018 Apr 2.

David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, USA; Department of Microbiology and Immunology, University of Rochester Medical Center, USA; Department of Pediatrics, Division of Infectious Diseases, University of Rochester Medical Center, USA.

CD4 T cells play a multiplicity of roles in protective immunity to influenza. Included in these functions are help for high affinity antibody production, enhancement of CD8 T cell expansion, function and memory, acceleration of the early innate response to infection and direct cytotoxicity. The influenza-specific CD4 T cell repertoire in humans established through exposures to infection and vaccination has been found to be highly variable in abundance, specificity and functionality. Deficits in particular subsets of CD4 T cells recruited into the response result in diminished antibody responses and protection from infection. Therefore, improved strategies for vaccination should include better methods to identify deficiencies in the circulating CD4 T cell repertoire, and vaccine constructs that increase the representation of CD4 T cells of the correct specificity and functionality.
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http://dx.doi.org/10.1016/j.coi.2018.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141328PMC
August 2018

Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains.

Proc Natl Acad Sci U S A 2017 11 6;114(47):12578-12583. Epub 2017 Nov 6.

Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;

H3N2 viruses continuously acquire mutations in the hemagglutinin (HA) glycoprotein that abrogate binding of human antibodies. During the 2014-2015 influenza season, clade 3C.2a H3N2 viruses possessing a new predicted glycosylation site in antigenic site B of HA emerged, and these viruses remain prevalent today. The 2016-2017 seasonal influenza vaccine was updated to include a clade 3C.2a H3N2 strain; however, the egg-adapted version of this viral strain lacks the new putative glycosylation site. Here, we biochemically demonstrate that the HA antigenic site B of circulating clade 3C.2a viruses is glycosylated. We show that antibodies elicited in ferrets and humans exposed to the egg-adapted 2016-2017 H3N2 vaccine strain poorly neutralize a glycosylated clade 3C.2a H3N2 virus. Importantly, antibodies elicited in ferrets infected with the current circulating H3N2 viral strain (that possesses the glycosylation site) and humans vaccinated with baculovirus-expressed H3 antigens (that possess the glycosylation site motif) were able to efficiently recognize a glycosylated clade 3C.2a H3N2 virus. We propose that differences in glycosylation between H3N2 egg-adapted vaccines and circulating strains likely contributed to reduced vaccine effectiveness during the 2016-2017 influenza season. Furthermore, our data suggest that influenza virus antigens prepared via systems not reliant on egg adaptations are more likely to elicit protective antibody responses that are not affected by glycosylation of antigenic site B of H3N2 HA.
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http://dx.doi.org/10.1073/pnas.1712377114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5703309PMC
November 2017

Pandemic 2009 H1N1 Influenza Venus reporter virus reveals broad diversity of MHC class II-positive antigen-bearing cells following infection in vivo.

Sci Rep 2017 09 7;7(1):10857. Epub 2017 Sep 7.

Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, NY, 14642, USA.

Although it is well established that Influenza A virus infection is initiated in the respiratory tract, the sequence of events and the cell types that become infected or access viral antigens remains incompletely understood. In this report, we used a novel Influenza A/California/04/09 (H1N1) reporter virus that stably expresses the Venus fluorescent protein to identify antigen-bearing cells over time in a mouse model of infection using flow cytometry. These studies revealed that many hematopoietic cells, including subsets of monocytes, macrophages, dendritic cells, neutrophils and eosinophils acquire influenza antigen in the lungs early post-infection. Surface staining of the viral HA revealed that most cell populations become infected, most prominently CD45 cells, alveolar macrophages and neutrophils. Finally, differences in infection status, cell lineage and MHC class II expression by antigen-bearing cells correlated with differences in their ability to re-stimulate influenza-specific CD4 T cells ex vivo. Collectively, these studies have revealed the cellular heterogeneity and complexity of antigen-bearing cells within the lung and their potential as targets of antigen recognition by CD4 T cells.
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http://dx.doi.org/10.1038/s41598-017-11313-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589842PMC
September 2017

Selective pre-priming of HA-specific CD4 T cells restores immunological reactivity to HA on heterosubtypic influenza infection.

PLoS One 2017 11;12(5):e0176407. Epub 2017 May 11.

Department of Pediatrics, Division of Infectious Diseases, University of Rochester Medical Center, Rochester, New York, United States of America.

A hallmark of the immune response to influenza is repeated encounters with proteins containing both genetically conserved and variable components. Therefore, the B and T cell repertoire is continually being remodeled, with competition between memory and naïve lymphocytes. Our previous work using a mouse model of secondary heterosubtypic influenza infection has shown that this competition results in a focusing of CD4 T cell response specificity towards internal virion proteins with a selective decrease in CD4 T cell reactivity to the novel HA epitopes. Strikingly, this shift in CD4 T cell specificity was associated with a diminished anti-HA antibody response. Here, we sought to determine whether the loss in HA-specific reactivity that occurs as a consequence of immunological memory could be reversed by selectively priming HA-specific CD4 T cells prior to secondary infection. Using a peptide-based priming strategy, we found that selective expansion of the anti-HA CD4 T cell memory repertoire enhanced HA-specific antibody production upon heterosubtypic infection. These results suggest that the potentially deleterious consequences of repeated exposure to conserved influenza internal virion proteins could be reversed by vaccination strategies that selectively arm the HA-specific CD4 T cell compartment. This could be a potentially useful pre-pandemic vaccination strategy to promote accelerated neutralizing antibody production on challenge with a pandemic influenza strain that contains few conserved HA epitopes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176407PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426616PMC
September 2017

Waning Immunity and Microbial Vaccines-Workshop of the National Institute of Allergy and Infectious Diseases.

Clin Vaccine Immunol 2017 Jul 5;24(7). Epub 2017 Jul 5.

Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA.

Since the middle of the 20th century, vaccines have made a significant public health impact by controlling infectious diseases globally. Although long-term protection has been achieved with some vaccines, immunity wanes over time with others, resulting in outbreaks or epidemics of infectious diseases. Long-term protection against infectious agents that have a complex life cycle and antigenic variation remains a key challenge. Novel strategies to characterize the short- and long-term immune responses to vaccines and to induce immune responses that mimic natural infection have recently emerged. New technologies and approaches in vaccinology, such as adjuvants, delivery systems, and antigen formulations, have the potential to elicit more durable protection and fewer adverse reactions; together with systems, these technologies have the capacity to model and accelerate vaccine development. The National Institute of Allergy and Infectious Diseases (NIAID) held a workshop on 19 September 2016 that focused on waning immunity to selected vaccines (for , serovar Typhi, , influenza, mumps, and malaria), with an emphasis on identifying knowledge gaps, future research needs, and how this information can inform development of more effective vaccines for infectious diseases.
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http://dx.doi.org/10.1128/CVI.00034-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5498725PMC
July 2017

Avian and Human Seasonal Influenza Hemagglutinin Proteins Elicit CD4 T Cell Responses That Are Comparable in Epitope Abundance and Diversity.

Clin Vaccine Immunol 2017 Mar 6;24(3). Epub 2017 Mar 6.

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, New York, USA

Avian influenza viruses remain a significant concern due to their pandemic potential. Vaccine trials have suggested that humans respond poorly to avian influenza vaccines relative to seasonal vaccines. It is important to understand, first, if there is a general deficiency in the ability of avian hemagglutinin (HA) proteins to generate immune responses and, if so, what underlies this defect. This question is of particular interest because it has been suggested that in humans, the poor immunogenicity of H7 vaccines may be due to a paucity of CD4 T cell epitopes. Because of the generally high levels of cross-reactive CD4 T cells in humans, it is not possible to compare the inherent immunogenicities of avian and seasonal HA proteins in an unbiased manner. Here, we empirically examine the epitope diversity and abundance of CD4 T cells elicited by seasonal and avian HA proteins. HLA-DR1 and HLA-DR4 transgenic mice were vaccinated with purified HA proteins, and CD4 T cells to specific epitopes were identified and quantified. These studies revealed that the diversity and abundance of CD4 T cells specific for HA do not segregate on the basis of whether the HA was derived from human seasonal or avian influenza viruses. Therefore, we conclude that failure in responses to avian vaccines in humans is likely due to a lack of cross-reactive CD4 T cell memory perhaps coupled with competition with or suppression of naive, HA-specific CD4 T cells by memory CD4 T cells specific for more highly conserved proteins.
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http://dx.doi.org/10.1128/CVI.00548-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339641PMC
March 2017

Nanoparticles decorated with viral antigens are more immunogenic at low surface density.

Vaccine 2017 02 3;35(5):774-781. Epub 2017 Jan 3.

Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642, United States. Electronic address:

There is an urgent need to develop protective vaccines for high priority viral pathogens. One approach known to enhance immune responses to viral proteins is to display them on a nanoparticle (NP) scaffold. However, little is known about the effect of protein density on the B cell response to antigens displayed on NPs. To address this question HIV-1 Envelope (Env) and influenza hemagglutinin (HA) were displayed on a polystyrene-based NP scaffold at various densities - corresponding to mean antigen distances that span the range encountered on naturally occurring virions. Our studies revealed that NPs displaying lower densities of Env or HA more efficiently stimulated antigen-specific B cells in vitro, as measured by calcium flux, than did NPs displaying higher antigen densities. Similarly, NPs displaying a low density of Env or HA also elicited higher titers of antigen-specific serum IgG in immunized BALB/c mice (including elevated titers of hemagglutination-inhibiting antibodies), as well as an increased frequency of antigen-specific antibody secreting cells in the lymph node, spleen and bone marrow. Importantly, our studies showed that the enhanced B cell response elicited by the lower density NPs is likely secondary to more efficient development of follicular helper CD4 T cells and germinal center B cells. These findings demonstrate that the density of antigen on a NP scaffold is a critical determinant of the humoral immune response elicited, and that high density display does not always result in an optimal response.
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http://dx.doi.org/10.1016/j.vaccine.2016.12.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5876043PMC
February 2017

Immunization with Pneumocystis Cross-Reactive Antigen 1 (Pca1) Protects Mice against Pneumocystis Pneumonia and Generates Antibody to Pneumocystis jirovecii.

Infect Immun 2017 04 23;85(4). Epub 2017 Mar 23.

Department of Pediatrics, University of Rochester, Rochester, New York, USA

pneumonia (PcP) is a life-threatening infection that affects immunocompromised individuals. Nearly half of all PcP cases occur in those prescribed effective chemoprophylaxis, suggesting that additional preventive methods are needed. To this end, we have identified a unique mouse surface protein, designated cross-reactive antigen 1 (Pca1), as a potential vaccine candidate. Mice were immunized with a recombinant fusion protein containing Pca1. Subsequently, CD4 T cells were depleted, and the mice were exposed to Pca1 immunization completely protected nearly all mice, similar to immunization with whole organisms. In contrast, all immunized negative-control mice developed PcP. Unexpectedly, Pca1 immunization generated cross-reactive antibody that recognized and Potential orthologs of Pca1 have been identified in Such cross-reactivity is rare, and our findings suggest that Pca1 is a conserved antigen and potential vaccine target. The evaluation of Pca1-elicited antibodies in the prevention of PcP in humans deserves further investigation.
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http://dx.doi.org/10.1128/IAI.00850-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364303PMC
April 2017

Flow Cytometric and Cytokine ELISpot Approaches To Characterize the Cell-Mediated Immune Response in Ferrets following Influenza Virus Infection.

J Virol 2016 09 12;90(17):7991-8004. Epub 2016 Aug 12.

Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center, Rochester, New York, USA

Unlabelled: Influenza virus infections represent a significant socioeconomic and public health burden worldwide. Although ferrets are considered by many to be ideal for modeling human responses to influenza infection and vaccination, efforts to understand the cellular immune response have been severely hampered by a paucity of standardized procedures and reagents. In this study, we developed flow cytometric and T cell enzyme-linked immunosorbent spot (ELISpot) approaches to characterize the leukocyte composition and antigen-specific T cell response within key lymphoid tissues following influenza virus infection in ferrets. Through a newly designed and implemented set of serological reagents, we used multiparameter flow cytometry to directly quantify the frequency of CD4(+) and CD8(+) T cells, Ig(+) B cells, CD11b(+) myeloid-derived cells, and major histocompatibility complex (MHC) class II-positive antigen-presenting cells (APCs) both prior to and after intranasal infection with A/California/04/09 (H1N1). We found that the leukocyte composition was altered at 10 days postinfection, with notable gains in the frequency of T cells and myeloid cells within the draining lymph node. Furthermore, these studies revealed that the antigen specificity of influenza virus-reactive CD4 and CD8 T cells was very broad, with recognition of the viral HA, NA, M1, NS1, and NP proteins, and that total reactivity to influenza virus postinfection represented approximately 0.1% of the circulating peripheral blood mononuclear cells (PBMC). Finally, we observed distinct patterns of reactivity between individual animals, suggesting heterogeneity at the MHC locus in ferrets within commercial populations, a finding of considerable interest in efforts to move the ferret model forward for influenza vaccine and challenge studies.

Importance: Ferrets are an ideal animal model to study transmission, diseases, and vaccine efficacies of respiratory viruses because of their close anatomical and physiological resemblances to humans. However, a lack of reagents has limited our understanding of the cell-mediated immune response following infection and vaccination. In this study, we used cross-reactive and ferret-specific antibodies to study the leukocyte composition and antigen-specific CD4 and CD8 T cell responses following influenza A/California/04/09 (H1N1) virus infection. These studies revealed strikingly distinct patterns of reactivity between CD4 and CD8 T cells, which were overlaid with differences in protein-specific responses between individual animals. Our results provide a first, in-depth look at the T cell repertoire in response to influenza infection and suggest that there is considerable heterogeneity at the MHC locus, which is akin to that in humans and an area of intense research interest.
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http://dx.doi.org/10.1128/JVI.01001-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988159PMC
September 2016

CD4 T cell epitope specificity determines follicular versus non-follicular helper differentiation in the polyclonal response to influenza infection or vaccination.

Sci Rep 2016 06 22;6:28287. Epub 2016 Jun 22.

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA.

Follicular helper T cells (Tfh) are essential for B cell production of high-affinity, class-switched antibodies. Much interest in Tfh development focuses on the priming environment of CD4 T cells. Here we explored the role that peptide specificity plays in the partitioning of the polyclonal CD4 T cell repertoire between Tfh and NonTfh lineages during the response to influenza. Surprisingly, we found that CD4 T cells specific for different epitopes exhibited distinct tendencies to segregate into Tfh or NonTfh. To alter the microenvironment and abundance, viral antigens were introduced as purified recombinant proteins in adjuvant as native proteins. Also, the most prototypical epitopes were expressed in a completely foreign protein. In many cases, the epitope-specific response patterns of Tfh vs. NonTfh persisted. The functional TcR avidity of only a subset of epitope-specific cells correlated with the tendency to drive a Tfh response. Thus, we conclude that in a polyclonal CD4 T cell repertoire, features of TcR-peptide:MHC class II complex have a strong deterministic influence on the ability of CD4 T cells to become a Tfh or a NonTfh. Our data is most consistent with at least 2 checkpoints of Tfh selection that include both TcR affinity and B cell presentation.
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http://dx.doi.org/10.1038/srep28287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4916409PMC
June 2016

The Role of CD4 T Cell Memory in Generating Protective Immunity to Novel and Potentially Pandemic Strains of Influenza.

Front Immunol 2016 25;7:10. Epub 2016 Jan 25.

Department of Microbiology and Immunology, David H. Smith Center for Vaccine Biology and Immunology, University of Rochester Medical Center , Rochester, NY , USA.

Recent events have made it clear that potentially pandemic strains of influenza regularly pose a threat to human populations. Therefore, it is essential that we develop better strategies to enhance vaccine design and evaluation to predict those that will be poor responders to vaccination and to identify those that are at particular risk of disease-associated complications following infection. Animal models have revealed the discrete functions that CD4 T cells play in developing immune response and to influenza immunity. However, humans have a complex immunological history with influenza through periodic infection and vaccination with seasonal variants, leading to the establishment of heterogeneous memory populations of CD4 T cells that participate in subsequent responses. The continual evolution of the influenza-specific CD4 T cell repertoire involves both specificity and function and overlays other restrictions on CD4 T cell activity derived from viral antigen handling and MHC class II:peptide epitope display. Together, these complexities in the influenza-specific CD4 T cell repertoire constitute a formidable obstacle to predicting protective immune response to potentially pandemic strains of influenza and in devising optimal vaccine strategies to potentiate these responses. We suggest that more precise efforts to identify and enumerate both the positive and negative contributors within the CD4 T cell compartment will aid significantly in the achievement of these goals.
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http://dx.doi.org/10.3389/fimmu.2016.00010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725218PMC
February 2016

Abundance and specificity of influenza reactive circulating memory follicular helper and non-follicular helper CD4 T cells in healthy adults.

Immunology 2015 Sep 14;146(1):157-62. Epub 2015 Jul 14.

David H. Smith Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, USA.

CD4 T-cell responses are functionally complex and regulate many aspects of innate and adaptive immunity. Follicular helper (Tfh) cells are CD4 T cells specialized to support B-cell production of isotype-switched, high-affinity antibody. So far, studies of Tfh cells in humans have focused on their differentiation requirements, with little research devoted to their antigen specificity. Here, after separating circulating human memory CD4 T cells based on expression of CXCR5, a signature marker of Tfh, we have quantified and assayed the influenza protein antigen specificity of blood Tfh cells and CD4 T cells lacking this marker. Through the use of peptide pools derived from nucleoprotein (NP) or haemagglutinin (HA) and a panel of human donors, we have discovered that circulating Tfh cells preferentially recognize peptide epitopes from HA while cells lacking CXCR5 are enriched for specificity toward NP. These studies suggest that reactive CD4 T cells specific for distinct viral antigens may have generalized differences in their functional potential due to their previous stimulation history.
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http://dx.doi.org/10.1111/imm.12491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4552510PMC
September 2015