Selected Publications
Click on each citation to read the abstract and reach links to the full publication.
Shehata L, Thouvenel CD, Hondowicz BD, Pew LA, Pritchard GH, Rawlings DJ, Choi J, Pepper M. (2024) Interleukin-4 downregulates transcription factor BCL6 to promote memory B cell selection in germinal centers. Immunity. PMID: 38513666
AbstractGerminal center (GC)-derived memory B cells (MBCs) are critical for humoral immunity as they differentiate into protective antibody-secreting cells during re-infection. GC formation and cellular interactions within the GC have been studied in detail, yet the exact signals that allow for the selection and exit of MBCs are not understood. Here, we showed that IL-4 cytokine signaling in GC B cells directly downregulated the transcription factor BCL6 via negative autoregulation to release cells from the GC program and to promote MBC formation. This selection event required additional survival cues and could therefore result in either GC exit or death. We demonstrate that both increasing IL-4 bioavailability or limiting IL-4 signaling disrupted MBC selection stringency. In this way, IL-4 control of BCL6 expression serves as a tunable switch within the GC to tightly regulate MBC selection and affinity maturation.
Rüterbusch MJ, Hondowicz BD, Takehara KK, Pruner KB, Griffith TS, Pepper M. (2023) Allergen exposure functionally alters influenza-specific CD4+ Th1 memory cells in the lung. J Exp Med. PMID: 37698553
Abstract CD4+ lung-resident memory T cells (TRM) generated in response to influenza infection confer effective protection against subsequent viral exposures. Whether these cells can be altered by environmental antigens and cytokines released during heterologous, antigen-independent immune responses is currently unclear. We therefore investigated how influenza-specific CD4+ Th1 TRM in the lung are impacted by a subsequent Th2-inducing respiratory house dust mite (HDM) exposure. Although naïve influenza-specific CD4+ T cells in the lymph nodes do not respond to HDM, influenza-specific CD4+ TRM in the lungs do respond to a subsequent allergen exposure by decreasing expression of the transcription factor T-bet. This functional alteration is associated with decreased IFN-γ production upon restimulation and improved disease outcomes following heterosubtypic influenza challenge. Further investigation revealed that ST2 signaling in CD4+ T cells during allergic challenge is necessary to induce these changes in lung-resident influenza-specific CD4+ TRM. Thus, heterologous antigen exposure or ST2-signaling can drive persistent changes in CD4+ Th1 TRM populations and impact protection upon reinfection.
Fontana MF, Ollmann Saphire E, Pepper M. (2023) Plasmodium infection disrupts the T follicular helper cell response to heterologous immunization. Elife. PMID: 36715223
Abstract Naturally acquired immunity to malaria develops only after many years and repeated exposures, raising the question of whether Plasmodium parasites, the etiological agents of malaria, suppress the ability of dendritic cells (DCs) to activate optimal T cell responses. We demonstrated recently that B cells, rather than DCs, are the principal activators of CD4+ T cells in murine malaria. In the present study, we further investigated factors that might prevent DCs from priming Plasmodium-specific T helper cell responses. We found that DCs were significantly less efficient at taking up infected red blood cells (iRBCs) compared to soluble antigen, whereas B cells more readily bound iRBCs. To assess whether DCs retained the capacity to present soluble antigen during malaria, we measured responses to a heterologous protein immunization administered to naïve mice or mice infected with P. chabaudi. Antigen uptake, DC activation, and expansion of immunogen-specific T cells were intact in infected mice, indicating DCs remained functional. However, polarization of the immunogen-specific response was dramatically altered, with a near-complete loss of germinal center T follicular helper cells specific for the immunogen, accompanied by significant reductions in antigen-specific B cells and antibody. Our results indicate that DCs remain competent to activate T cells during Plasmodium infection, but that T cell polarization and humoral responses are severely disrupted. This study provides mechanistic insight into the development of both Plasmodium-specific and heterologous adaptive responses in hosts with malaria.
Hale M, Netland J, Chen Y, Thouvenel CD, Smith KN, Rich LM, Vanderwall ER, Miranda MC, Eggenberger J, Hao L, Watson MJ, Mundorff CC, Rodda LB, King NP, Guttman M, Gale M, Abraham J, Debley JS, Pepper M, Rawlings DJ. (2022) IgM antibodies derived from memory B cells are potent cross-variant neutralizers of SARS-CoV-2. J Exp Med. PMID: 35938988
Abstract Humoral immunity to SARS-CoV-2 can be supplemented with polyclonal sera from convalescent donors or an engineered monoclonal antibody (mAb) product. While pentameric IgM antibodies are responsible for much of convalescent sera's neutralizing capacity, all available mAbs are based on the monomeric IgG antibody subtype. We now show that IgM mAbs derived from immune memory B cell receptors are potent neutralizers of SARS-CoV-2. IgM mAbs outperformed clonally identical IgG antibodies across a range of affinities and SARS-CoV-2 receptor-binding domain epitopes. Strikingly, efficacy against SARS-CoV-2 viral variants was retained for IgM but not for clonally identical IgG. To investigate the biological role for IgM memory in SARS-CoV-2, we also generated IgM mAbs from antigen-experienced IgM+ memory B cells in convalescent donors, identifying a potent neutralizing antibody. Our results highlight the therapeutic potential of IgM mAbs and inform our understanding of the role for IgM memory against a rapidly mutating pathogen.
Rodda LB, Morawski PA, Pruner KB, Fahning ML, Howard CA, Franko N, Logue J, Eggenberger J, Stokes C, Golez I, Hale M, Gale Jr M, Chu HY, Campbell DJ, Pepper M. (2022) Imprinted SARS-CoV-2-specific memory lymphocytes define hybrid immunity. Cell. PMID: 35413241
Abstract Immune memory is tailored by cues that lymphocytes perceive during priming. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic created a situation in which nascent memory could be tracked through additional antigen exposures. Both SARS-CoV-2 infection and vaccination induce multifaceted, functional immune memory, but together, they engender improved protection from disease, termed hybrid immunity. We therefore investigated how vaccine-induced memory is shaped by previous infection. We found that following vaccination, previously infected individuals generated more SARS-CoV-2 RBD-specific memory B cells and variant-neutralizing antibodies and a distinct population of IFN-γ and IL-10-expressing memory SARS-CoV-2 spike-specific CD4+ T cells than previously naive individuals. Although additional vaccination could increase humoral memory in previously naive individuals, it did not recapitulate the distinct CD4+ T cell cytokine profile observed in previously infected subjects. Thus, imprinted features of SARS-CoV-2-specific memory lymphocytes define hybrid immunity.
Rodda LB, Netland J, Shehata L, Pruner KB, Morawski PA, Thouvenel CD, Takehara KK, Eggenberger J, Hemann EA, Waterman HR, Fahning ML, Chen Y, Hale M, Rathe J, Stokes C, Wrenn S, Fiala B, Carter L, Hamerman JA, King NP, Gale Jr M, Campbell DJ, Rawlings DJ, Pepper M. (2021) Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19. Cell. PMID: 33296701
Abstract The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is causing a global pandemic, and cases continue to rise. Most infected individuals experience mildly symptomatic coronavirus disease 2019 (COVID-19), but it is unknown whether this can induce persistent immune memory that could contribute to immunity. We performed a longitudinal assessment of individuals recovered from mild COVID-19 to determine whether they develop and sustain multifaceted SARS-CoV-2-specific immunological memory. Recovered individuals developed SARS-CoV-2-specific immunoglobulin (IgG) antibodies, neutralizing plasma, and memory B and memory T cells that persisted for at least 3 months. Our data further reveal that SARS-CoV-2-specific IgG memory B cells increased over time. Additionally, SARS-CoV-2-specific memory lymphocytes exhibited characteristics associated with potent antiviral function: memory T cells secreted cytokines and expanded upon antigen re-encounter, whereas memory B cells expressed receptors capable of neutralizing virus when expressed as monoclonal antibodies. Therefore, mild COVID-19 elicits memory lymphocytes that persist and display functional hallmarks of antiviral immunity.
Arroyo EN, Pepper M. (2020) B cells are sufficient to prime the dominant CD4+ Tfh response to Plasmodium infection. J Exp Med. PMID: 31748243
Abstract CD4+ T follicular helper (Tfh) cells dominate the acute response to a blood-stage Plasmodium infection and provide signals to direct B cell differentiation and protective antibody expression. We studied antigen-specific CD4+ Tfh cells responding to Plasmodium infection in order to understand the generation and maintenance of the Tfh response. We discovered that a dominant, phenotypically stable, CXCR5+ Tfh population emerges within the first 4 d of infection and results in a CXCR5+ CCR7+ Tfh/central memory T cell response that persists well after parasite clearance. We also found that CD4+ T cell priming by B cells was both necessary and sufficient to generate this Tfh-dominant response, whereas priming by conventional dendritic cells was dispensable. This study provides important insights into the development of CD4+ Tfh cells during Plasmodium infection and highlights the heterogeneity of antigen-presenting cells involved in CD4+ T cell priming.
Hondowicz BD, Kim KS, Ruterbusch MJ, Keitany GJ, Pepper M. (2018) IL-2 is required for the generation of viral-specific CD4+ Th1 tissue-resident memory cells and B cells are essential for maintenance in the lung. Eur J Immunol. PMID: 28948612
Abstract CD4+ tissue resident cells are an important first line of defense against viral infections in the lungs and are critical for promoting the localization of lung resident CD8+ T cells. However, relatively little is known about the signaling programs required for the development of viral-specific CD4+ tissue resident cells in the lungs. Recently, it was shown that signaling through the high affinity IL-2 receptor is required for the differentiation of lung-resident Th2 memory (Trm) cells in a murine model of airway inflammation. We therefore tested if IL-2 signaling is also required for the development of viral antigen-specific CD4+ Th1 cells in the lung after i.n. infection with lymphocytic choriomeningitis virus. These studies demonstrate that Th1 CD4+ T cells also require IL-2 for lung Trm development. Additionally, they show that B cells potently inhibit early Th1 cell lung residency, but are required for the maintenance of a long-lived population of CD4+Th1 Trm.
Keitany GJ, Kim KS, Krishnamurty AT, Hondowicz BD, Hahn WO, Dambrauskas N, Sather DN, Vaughan AM, Kappe SH, Pepper M. (2016) Blood Stage Malaria Disrupts Humoral Immunity to the Pre-erythrocytic Stage Circumsporozoite Protein. Cell Reports. PMID: 28009289
Abstract Many current malaria vaccines target the pre-erythrocytic stage of infection in the liver. However, in malaria-endemic regions, increased blood stage exposure is associated with decreased vaccine efficacy, thereby challenging current vaccine efforts. We hypothesized that pre-erythrocytic humoral immunity is directly disrupted by blood stage infection. To investigate this possibility, we used Plasmodium-antigen tetramers to analyze B cells after infection with either late liver stage arresting parasites or wild-type parasites that progress to the blood stage. Our data demonstrate that immunoglobulin G (IgG) antibodies against the pre-erythrocytic antigen, circumsporozoite protein (CSP), are generated only in response to the attenuated, but not the wild-type, infection. Further analyses revealed that blood stage malaria inhibits CSP-specific germinal center B cell differentiation and modulates chemokine expression. This results in aberrant memory formation and the loss of a rapid secondary B cell response. These data highlight how immunization with attenuated parasites may drive optimal immunity to malaria.
Krishnamurty AT, Thouvenel CD, Portugal S, Keitany GJ, Kim KS, Holder A, Crompton PD, Rawlings DJ, Pepper M. (2016) Somatically Hypermutated Plasmodium-Specific IgM(+) Memory B Cells Are Rapid, Plastic, Early Responders Upon Malaria Rechallenge. Immunity. PMID: 27473412
Abstract Humoral immunity consists of pre-existing antibodies expressed by long-lived plasma cells and rapidly reactive memory B cells (MBC). Recent studies of MBC development and function after protein immunization have uncovered significant MBC heterogeneity. To clarify functional roles for distinct MBC subsets during malaria infection, we generated tetramers that identify Plasmodium-specific MBCs in both humans and mice. Long-lived murine Plasmodium-specific MBCs consisted of three populations: somatically hypermutated immunoglobulin M(+) (IgM(+)) and IgG(+) MBC subsets and an unmutated IgD(+) MBC population. Rechallenge experiments revealed that high affinity, somatically hypermutated Plasmodium-specific IgM(+) MBCs proliferated and gave rise to antibody-secreting cells that dominated the early secondary response to parasite rechallenge. IgM(+) MBCs also gave rise to T cell-dependent IgM(+) and IgG(+)B220(+)CD138(+) plasmablasts or T cell-independent B220(-)CD138(+) IgM(+) plasma cells. Thus, even in competition with IgG(+) MBCs, IgM(+) MBCs are rapid, plastic, early responders to a secondary Plasmodium rechallenge and should be targeted by vaccine strategies.
Hondowicz BD, An D, Schenkel JM, Kim KS, Steach HR, Krishnamurty AT, Keitany GJ, Garza EN, Fraser KA, Moon JJ, Altemeier WA, Masopust D, Pepper M. (2016) Interleukin-2-Dependent Allergen-Specific Tissue-Resident Memory Cells Drive Asthma. Immunity. PMID: 26750312
Abstract Exposure to inhaled allergens generates T helper 2 (Th2) CD4(+) T cells that contribute to episodes of inflammation associated with asthma. Little is known about allergen-specific Th2 memory cells and their contribution to airway inflammation. We generated reagents to understand how endogenous CD4(+) T cells specific for a house dust mite (HDM) allergen form and function. After allergen exposure, HDM-specific memory cells persisted as central memory cells in the lymphoid organs and tissue-resident memory cells in the lung. Experimental blockade of lymphocyte migration demonstrated that lung-resident cells were sufficient to induce airway hyper-responsiveness, which depended upon CD4(+) T cells. Investigation into the differentiation of pathogenic Trm cells revealed that interleukin-2 (IL-2) signaling was required for residency and directed a program of tissue homing migrational cues. These studies thus identify IL-2-dependent resident Th2 memory cells as drivers of lung allergic responses.
Pepper M, Pagán AJ, Igyártó BZ,Taylor JJ and Jenkins MK. (2011) Opposing signals from the Bcl6 transcription factor and the interleukin-2 receptor generate T helper-1 central and effector memory cells. Immunity. 35 (4): 583-595. PMID: 22018468.
Abstract Listeria monocytogenes infection generates T helper 1 (Th1) effector memory cells and CC chemokine receptor 7 (CCR7)(+) cells resembling central memory cells. We tracked endogenous L. monocytogenes-specific CD4(+) T cells to determine how these memory cells are formed. Two effector cell populations were already present several days after infection. One highly expressed the T-bet transcription factor and produced Th1 memory cells in an interleukin-2 (IL-2) receptor-dependent fashion. The other resided in the T cell areas, expressed CCR7 and CXC chemokine receptor 5 (CXCR5), and like follicular helper cells depended on the Bcl6 transcription factor and inducible costimulator ligand on B cells. The CCR7(+)CXCR5(+) effector cells produced similar memory cells that generated diverse effector cell populations in a secondary response. Thus, Th1 effector memory and follicular helper-like central memory cells are produced from early effector cell populations that diverge in response to signals from the IL-2 receptor, Bcl6, and B cells.
Pepper M and Marc K. Jenkins. (2011) Origins of CD4+ Effector and Central Memory T cells. Nature Immunology. 131(6): 467-71. PMID: 21739668
Abstract Lineage-committed effector CD4(+) T cells are generated at the peak of the primary response and are followed by heterogeneous populations of central and effector memory cells. Here we review the evidence that T helper type 1 (T(H)1) effector cells survive the contraction phase of the primary response and become effector memory cells. We discuss the applicability of this idea to the T(H)2 cell, T(H)17 helper T cell, follicular helper T cell (T(FH) cell) and induced regulatory T cell lineages. We also discuss how central memory cells are formed, with an emphasis on the role of B cells in this process.
Moon JJ, Chu HH, Hataye J, Pagán AJ, Pepper M, McLachlan JB, Zell T, and Jenkins MK. (2009) Tracking epitope-specific T cells. Nature Protocols 4:565-581. PMID: 19373228
Abstract The tracking of antigen-specific T cells in vivo is a useful approach for the study of the adaptive immune response. This protocol describes how populations of T cells specific for a given peptide-major histocompatibility complex (pMHC) epitope can be tracked based solely on T-cell receptor (TCR) specificity as opposed to other indirect methods based on function. The methodology involves the adoptive transfer of TCR transgenic T cells with defined epitope specificity into histocompatible mice and the subsequent detection of these cells through the use of congenic or clonotypic markers. Alternatively, endogenous epitope-specific T cells can be tracked directly through the use of pMHC tetramers. Using magnetic bead-based enrichment and advanced multiparameter flow cytometry, populations as small as five epitope-specific T cells can be detected from the peripheral lymphoid organs of a mouse. The adoptive transfer procedure can be completed within 3 h, whereas analysis of epitope-specific cells from mice can be completed within 6 h.
Moon, J.J., Chu, H.H., Pepper, M., McSorley, S.J., Jameson, S.C., Kedl, RM, Jenkins, MK. Naive CD4+ T cell population size predicts immune response magnitude and diversity. (2007) Naïve CD4+ T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude. Immunity. 27 (2):203-213. PMID: 17707129
Abstract Cell-mediated immunity stems from the proliferation of naive T lymphocytes expressing T cell antigen receptors (TCRs) specific for foreign peptides bound to host major histocompatibility complex (MHC) molecules. Because of the tremendous diversity of the T cellrepertoire, naive T cells specific for any one peptide:MHC complex (pMHC) are extremely rare. Thus, it is not known how many naive T cells of any given pMHC specificity exist in the body or how that number influences the immune response. By using soluble pMHC class II (pMHCII) tetramers and magnetic bead enrichment, we found that three different pMHCII-specific naive CD4(+) T cell populations vary in frequency from 20 to 200 cells per mouse. Moreover, naive population size predicted the size and TCR diversity of the primary CD4(+) T cell response after immunization with relevant peptide. Thus, variation in naive T cell frequencies can explain why some peptides are stronger immunogens than others.
Pepper, M., Dzierszinski, F.,Crawford, A., Hunter, C., Roos, D. (2004) Development of a system to study CD4+-T-cell responses to transgenic ovalbumin-expressing Toxoplasma gondii during toxoplasmosis. Infection and Immunity. 72(12): 7240-6. PMID: 15557649
The study of the immune response to Toxoplasma gondii has provided numerous insights into the role of T cells in resistance to intracellular infections. However, the complexity of this eukaryote pathogen has made it difficult to characterize immunodominant epitopes that would allow the identification of T cells with a known specificity for parasite antigens. As a consequence, analysis of T-cell responses to T. gondii has been based on characterization of the percentage of T cells that express an activated phenotype during infection and on the ability of these cells to produce cytokines in response to complex mixtures of parasite antigens. In order to study specific CD4(+) T cells responses to T. gondii, recombinant parasites that express a truncated ovalbumin (OVA) protein, in either a cytosolic or a secreted form, were engineered. In vitro and in vivo studies reveal that transgenic parasites expressing secreted OVA are able to stimulate T-cell receptor-transgenic OVA-specific CD4(+) T cells to proliferate, express an activated phenotype, and produce gamma interferon (IFN-gamma). Furthermore, the adoptive transfer of OVA-specific T cells into IFN-gamma(-/-) mice provided enhanced protection against infection with the OVA-transgenic (but not parental) parasites. Together, these studies establish the utility of this transgenic system to study CD4(+)-T-cell responses during toxoplasmosis.
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