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1. 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
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.
2. 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
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.
3. 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
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.
4. 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
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.
5. 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.
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 memorycells 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 Bcl6transcription 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 centralmemory cells are produced from early effector cell populations that diverge in response to signals from the IL-2 receptor, Bcl6, and B cells.
6. Pepper M and Marc K. Jenkins. (2011) Origins of CD4+ Effector and Central Memory T cells. Nature Immunology. 131(6): 467-71. PMID: 21739668
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.
7. 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
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.
8. 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
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.
9. 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.