Bali Pulendran

Publications of Bali Pulendran

• Distinct TLR adjuvants differentially stimulate systemic and local innate immune responses in nonhuman primates.

  Authors: Marcin Kwissa, Helder I Nakaya, Herold Oluoch, Bali Pulendran

  Blood. 03/2012; 119(9):2044-55.

  TLR ligands (TLR-Ls) represent novel vaccine adjuvants, but their immunologic effects in humans remain poorly defined in vivo. In the present study, we analyzed the innate responses stimulated byTLR ligands (TLR-Ls) represent novel vaccine adjuvants, but their immunologic effects in humans remain poorly defined in vivo. In the present study, we analyzed the innate responses stimulated by different TLR-Ls in rhesus macaques. MPL (TLR4-L), R-848 (TLR7/8-L), or cytosine-phosphate-guanine oligodeoxynucleotide (TLR9-L) induced a rapid and robust expansion of blood neutrophils, with a concomitant reduction in PBMCs. Furthermore, all TLR-Ls induced rapid (3-8 hours) expansion of CD14(+) monocytes, but only TLR7/8-L and TLR9-L mobilized the CD14(+)CD16(+) and CD14(dim)CD16(++) monocytes, and only TLR7/8-L and TLR9-L induced activation of myeloid dendritic cells (mDCs) and plasmacytoid DCs (pDCs), production of IP-10 and type-I IFN, and expression of type-I IFN-related and chemokine genes in the blood. In the draining lymph nodes (LNs), consistent with the effects in blood, all TLR-Ls induced expansion of CD14(+) monocytes, but only TLR7/8-L and TLR9-L expanded the activated CD14(+)CD16(+) cells. TLR4-L and TLR9-L differentially induced the expansion of mDCs and pDCs (1-3 days), but did not activate DCs. In contrast, TLR7/8-L did not induce DC expansion, but did activate mDCs. Finally, both TLR9-L and TLR7/8-L induced the expression of genes related to chemokines and type-I IFNs in LNs. Thus different TLR-Ls mediate distinct signatures of early innate responses both locally and systemically.

• Rapid and Massive Virus-Specific Plasmablast Responses during Acute Dengue Virus Infection in Humans.

  Authors: Jens Wrammert, Nattawat Onlamoon, Rama S Akondy, Guey C Perng, Korakot Polsrila, Anmol Chandele, Marcin Kwissa, Bali Pulendran, Patrick C Wilson, Orasri Wittawatmongkol, Sutee Yoksan, Nasikarn Angkasekwinai, Kovit Pattanapanyasat, Kulkanya Chokephaibulkit, Rafi Ahmed

  Journal of virology. 03/2012; 86(6):2911-8.

  Humoral immune responses are thought to play a major role in dengue virus-induced immunopathology; however, little is known about the plasmablasts producing these antibodies during an ongoingHumoral immune responses are thought to play a major role in dengue virus-induced immunopathology; however, little is known about the plasmablasts producing these antibodies during an ongoing infection. Herein we present an analysis of plasmablast responses in patients with acute dengue virus infection. We found very potent plasmablast responses that often increased more than 1,000-fold over the baseline levels in healthy volunteers. In many patients, these responses made up as much 30% of the peripheral lymphocyte population. These responses were largely dengue virus specific and almost entirely made up of IgG-secreting cells, and plasmablasts reached very high numbers at a time after fever onset that generally coincided with the window where the most serious dengue virus-induced pathology is observed. The presence of these large, rapid, and virus-specific plasmablast responses raises the question as to whether these cells might have a role in dengue immunopathology during the ongoing infection. These findings clearly illustrate the need for a detailed understanding of the repertoire and specificity of the antibodies that these plasmablasts produce.

• Systems vaccinology: learning to compute the behavior of vaccine induced immunity.

  Authors: Helder I Nakaya, Shuzhao Li, Bali Pulendran

  Wiley interdisciplinary reviews. Systems biology and medicine. 03/2012; 4(2):193-205.

  The goal of systems biology is to access and integrate information about the parts (e.g., genes, proteins, cells) of a biological system with a view to computing and predicting the behavior of theThe goal of systems biology is to access and integrate information about the parts (e.g., genes, proteins, cells) of a biological system with a view to computing and predicting the behavior of the system. The past decade has witnessed technological revolutions in the capacity to make high throughput measurements about the behavior of genes, proteins, and cells. Such technologies are widely used in biological research and in medicine, such as toward prognosis and therapy response prediction in cancer patients. More recently, systems biology is being applied to vaccinology, with the goal of: (1) understanding the mechanisms by which vaccines stimulate protective immunity, and (2) predicting the immunogenicity or efficacy of vaccines. Here, we review the recent advances in this area, and highlight the biological and computational challenges posed. WIREs Syst Biol Med 2012, 4:193-205. doi: 10.1002/wsbm.163 For further resources related to this article, please visit the WIREs website.

• Systems biology in understanding HIV pathogenesis and guiding vaccine development.

  Authors: Rafick Sekaly, Bali Pulendran

  Current opinion in HIV and AIDS. 11/2011; 7(1):1-3.

• Learning vaccinology from viral infections.

  Authors: Rafi Ahmed, Bali Pulendran

  The Journal of experimental medicine. 11/2011; 208(12):2347-9.

  This issue of the Journal of Experimental Medicine celebrates and honors the life of Ralph Steinman (1943-2011), winner of the 2011 Nobel Prize in Physiology or Medicine. Ralph's science was rootedThis issue of the Journal of Experimental Medicine celebrates and honors the life of Ralph Steinman (1943-2011), winner of the 2011 Nobel Prize in Physiology or Medicine. Ralph's science was rooted in fundamental discovery with the goal of translating these findings into clinical medicine. He recognized the power of immunology in treating human disease and passionately championed studies on vaccine design, immune therapy, and human immunology. One particular collaborative effort between the Steinman and Sekaly laboratories resulted in a paper published in this issue of the journal.

• An open-ended plea for the development of a global database of HIV vaccine responses.

  Authors: Peter Wilkinson, Abdelali Filali-Mouhim, Shuzhao Li, Jeffrey Ahlers, John Schatzle, Bali Pulendran, Rafick-Pierre Sekaly, Mark J Cameron

  Current opinion in HIV and AIDS. 11/2011; 7(1):10-6.

  The purpose of this review is to describe a critical need of the HIV research community for a globally accessible database of HIV vaccine responses that stores data from multiple assay platforms inThe purpose of this review is to describe a critical need of the HIV research community for a globally accessible database of HIV vaccine responses that stores data from multiple assay platforms in the form of lists of correlates of immune protection and vaccine efficacy. This is not a detailed review but a first step toward developing a dialogue among investigators and funding organizations to build upon existing resources to efficiently develop a HIV vaccine response and correlates database. We also discuss examples of databases that complement our needs and could be integrated into our proposed database requirements. Several vaccine-related databases that store information at the study level currently exist, however, at the present time, a correlates of immune protection database does not exist. Here, we discuss the scientific climate surrounding HIV vaccine development with the evolution of systems biology approaches, the problems at hand for analyzing and harmonizing datasets generated from preclinical and clinical studies, and the curation and accessibility of useful information to model outcomes. We also compare key database requirements of a few existing globally accessible databases and provide several illustrative correlate database submission and utilization examples.

• Systems vaccinology: its promise and challenge for HIV vaccine development.

  Authors: Helder I Nakaya, Bali Pulendran

  Current opinion in HIV and AIDS. 11/2011; 7(1):24-31.

  The use of systems biology approaches to understand and predict vaccine-induced immunity promises to revolutionize vaccinology. For centuries vaccines were developed empirically, with very littleThe use of systems biology approaches to understand and predict vaccine-induced immunity promises to revolutionize vaccinology. For centuries vaccines were developed empirically, with very little understanding of the mechanisms by which they mediate protective immunity. The so-called systems vaccinology approach employs high-throughput technologies (e.g. microarrays, RNA-seq and mass spectrometry-based proteomics and metabolomics) and computational modeling to describe the complex interactions between all the parts of immune system, with a view to elucidating new biological rules capable of predicting the behavior of the system. Systems biology successfully applied to yellow-fever and influenza vaccines has led to the discovery of signatures that predict vaccine immunogenicity, and promises to advance basic immunology research by providing novel mechanistic insights about immune regulation. However a major challenge of systems vaccinology concerns the analyses and interpretation of the large and noisy data sets generated by high-throughput techniques. Overcoming these issues, we envision that systems vaccinology will have a potential impact on vaccine development, including HIV vaccines. High-throughput technologies allow the investigation of vaccine-induced immune responses at system and molecular levels. These are currently being used to unravel new molecular insights about the immune system, and are on the verge of being integrated into clinical trials to enable rational vaccine design and development.

• Functional specializations of intestinal dendritic cell and macrophage subsets that control Th17 and regulatory T cell responses are dependent on the T cell/APC ratio, source of mouse strain, and regional localization.

  Authors: Timothy L Denning, Brian A Norris, Oscar Medina-Contreras, Santhakumar Manicassamy, Duke Geem, Rajat Madan, Christopher L Karp, Bali Pulendran

  Journal of immunology (Baltimore, Md. : 1950). 06/2011; 187(2):733-47.

  Although several subsets of intestinal APCs have been described, there has been no systematic evaluation of their phenotypes, functions, and regional localization to date. In this article, we usedAlthough several subsets of intestinal APCs have been described, there has been no systematic evaluation of their phenotypes, functions, and regional localization to date. In this article, we used 10-color flow cytometry to define the major APC subsets in the small and large intestine lamina propria. Lamina propria APCs could be subdivided into CD11c(+)CD11b(-), CD11c(+)CD11b(+), and CD11c(dull)CD11b(+) subsets. CD11c(+)CD11b(-) cells were largely CD103(+)F4/80(-) dendritic cells (DCs), whereas the CD11c(+)CD11b(+) subset comprised CD11c(+)CD11b(+)CD103(+)F4/80(-) DCs and CD11c(+)CD11b(+)CD103(-)F4/80(+) macrophage-like cells. The majority of CD11c(dull)CD11b(+) cells were CD103(-)F4/80(+) macrophages. Although macrophages were more efficient at inducing Foxp3(+) regulatory T (T(reg)) cells than DCs, at higher T cell/APC ratios, all of the DC subsets efficiently induced Foxp3(+) T(reg) cells. In contrast, only CD11c(+)CD11b(+)CD103(+) DCs efficiently induced Th17 cells. Consistent with this, the regional distribution of CD11c(+)CD11b(+)CD103(+) DCs correlated with that of Th17 cells, with duodenum > jejunum > ileum > colon. Conversely, CD11c(+)CD11b(-)CD103(+) DCs, macrophages, and Foxp3(+) T(reg) cells were most abundant in the colon and scarce in the duodenum. Importantly, however, the ability of DC and macrophage subsets to induce Foxp3(+) T(reg) cells versus Th17 cells was strikingly dependent on the source of the mouse strain. Thus, DCs from C57BL/6 mice from Charles River Laboratories (that have segmented filamentous bacteria, which induce robust levels of Th17 cells in situ) were more efficient at inducing Th17 cells and less efficient at inducing Foxp3(+) T(reg) cells than DCs from B6 mice from The Jackson Laboratory. Thus, the functional specializations of APC subsets in the intestine are dependent on the T cell/APC ratio, regional localization, and source of the mouse strain.

• Immunological mechanisms of vaccination.

  Authors: Bali Pulendran, Rafi Ahmed

  Nature immunology. 06/2011; 12(6):509-17.

  Vaccines represent one of the greatest triumphs of modern medicine. Despite the common origins of vaccinology and immunology more than 200 years ago, the two disciplines have evolved along suchVaccines represent one of the greatest triumphs of modern medicine. Despite the common origins of vaccinology and immunology more than 200 years ago, the two disciplines have evolved along such different trajectories that most of the highly successful vaccines have been made empirically, with little or no immunological insight. Recent advances in innate immunity have offered new insights about the mechanisms of vaccine-induced immunity and have facilitated a more rational approach to vaccine design. Here we will discuss these advances and emerging themes on the immunology of vaccination.

• Dendritic cell control of tolerogenic responses.

  Authors: Santhakumar Manicassamy, Bali Pulendran

  Immunological reviews. 05/2011; 241(1):206-27.

  One of the most fundamental problems in immunology is the seemingly schizophrenic ability of the immune system to launch robust immunity against pathogens, while acquiring and maintaining a state ofOne of the most fundamental problems in immunology is the seemingly schizophrenic ability of the immune system to launch robust immunity against pathogens, while acquiring and maintaining a state of tolerance to the body's own tissues and the trillions of commensal microorganisms and food antigens that confront it every day. A fundamental role for the innate immune system, particularly dendritic cells (DCs), in orchestrating immunological tolerance has been appreciated, but emerging studies have highlighted the nature of the innate receptors and the signaling pathways that program DCs to a tolerogenic state. Furthermore, several studies have emphasized the major role played by cellular interactions and the microenvironment in programming tolerogenic DCs. Here, we review these studies and suggest that the innate control of tolerogenic responses can be viewed as different hierarchies of organization, in which DCs, their innate receptors and signaling networks, and their interactions with other cells and local microenvironments represent different levels of the hierarchy.

• Phenotype, function, and gene expression profiles of programmed death-1(hi) CD8 T cells in healthy human adults.

  Authors: Jaikumar Duraiswamy, Chris C Ibegbu, David Masopust, Joseph D Miller, Koichi Araki, Gregory H Doho, Pramila Tata, Satish Gupta, Michael J Zilliox, Helder I Nakaya, Bali Pulendran, W Nicholas Haining, Gordon J Freeman, Rafi Ahmed

  Journal of immunology (Baltimore, Md. : 1950). 03/2011; 186(7):4200-12.

  T cell dysfunction is an important feature of many chronic viral infections. In particular, it was shown that programmed death-1 (PD-1) regulates T cell dysfunction during chronic lymphocyticT cell dysfunction is an important feature of many chronic viral infections. In particular, it was shown that programmed death-1 (PD-1) regulates T cell dysfunction during chronic lymphocytic choriomeningitis virus infection in mice, and PD-1(hi) cells exhibit an intense exhausted gene signature. These findings were extended to human chronic infections such as HIV, hepatitis C virus, and hepatitis B virus. However, it is not known if PD-1(hi) cells of healthy humans have the traits of exhausted cells. In this study, we provide a comprehensive description of phenotype, function, and gene expression profiles of PD-1(hi) versus PD-1(lo) CD8 T cells in the peripheral blood of healthy human adults as follows: 1) the percentage of naive and memory CD8 T cells varied widely in the peripheral blood cells of healthy humans, and PD-1 was expressed by the memory CD8 T cells; 2) PD-1(hi) CD8 T cells in healthy humans did not significantly correlate with the PD-1(hi) exhausted gene signature of HIV-specific human CD8 T cells or chronic lymphocytic choriomeningitis virus-specific CD8 T cells from mice; 3) PD-1 expression did not directly affect the ability of CD8 T cells to secrete cytokines in healthy adults; 4) PD-1 was expressed by the effector memory compared with terminally differentiated effector CD8 T cells; and 5) finally, an interesting inverse relationship between CD45RA and PD-1 expression was observed. In conclusion, our study shows that most PD-1(hi) CD8 T cells in healthy adult humans are effector memory cells rather than exhausted cells.

• Programming the magnitude and persistence of antibody responses with innate immunity.

  Authors: Sudhir Pai Kasturi, Ioanna Skountzou, Randy A Albrecht, Dimitrios Koutsonanos, Tang Hua, Helder I Nakaya, Rajesh Ravindran, Shelley Stewart, Munir Alam, Marcin Kwissa, Francois Villinger, Niren Murthy, John Steel, Joshy Jacob, Robert J Hogan, Adolfo García-Sastre, Richard Compans, Bali Pulendran

  Nature. 02/2011; 470(7335):543-7.

  Many successful vaccines induce persistent antibody responses that can last a lifetime. The mechanisms by which they do so remain unclear, but emerging evidence indicates that they activate dendriticMany successful vaccines induce persistent antibody responses that can last a lifetime. The mechanisms by which they do so remain unclear, but emerging evidence indicates that they activate dendritic cells via Toll-like receptors (TLRs). For example, the yellow fever vaccine YF-17D, one of the most successful empiric vaccines ever developed, activates dendritic cells via multiple TLRs to stimulate proinflammatory cytokines. Triggering specific combinations of TLRs in dendritic cells can induce synergistic production of cytokines, which results in enhanced T-cell responses, but its impact on antibody responses remain unknown. Learning the critical parameters of innate immunity that program such antibody responses remains a major challenge in vaccinology. Here we demonstrate that immunization of mice with synthetic nanoparticles containing antigens plus ligands that signal through TLR4 and TLR7 induces synergistic increases in antigen-specific, neutralizing antibodies compared to immunization with nanoparticles containing antigens plus a single TLR ligand. Consistent with this there was enhanced persistence of germinal centres and of plasma-cell responses, which persisted in the lymph nodes for >1.5 years. Surprisingly, there was no enhancement of the early short-lived plasma-cell response relative to that observed with single TLR ligands. Molecular profiling of activated B cells, isolated 7 days after immunization, indicated that there was early programming towards B-cell memory. Antibody responses were dependent on direct triggering of both TLRs on B cells and dendritic cells, as well as on T-cell help. Immunization protected completely against lethal avian and swine influenza virus strains in mice, and induced robust immunity against pandemic H1N1 influenza in rhesus macaques.

• Systems biology of vaccination for seasonal influenza in humans.

  Authors: Helder I Nakaya, Jens Wrammert, Eva K Lee, Luigi Racioppi, Stephanie Marie-Kunze, W Nicholas Haining, Anthony R Means, Sudhir P Kasturi, Nooruddin Khan, Gui-Mei Li, Megan McCausland, Vibhu Kanchan, Kenneth E Kokko, Shuzhao Li, Rivka Elbein, Aneesh K Mehta, Alan Aderem, Kanta Subbarao, Rafi Ahmed, Bali Pulendran

  Nature immunology. 01/2011; 12(8):786-95.

  Here we have used a systems biology approach to study innate and adaptive responses to vaccination against influenza in humans during three consecutive influenza seasons. We studied healthy adultsHere we have used a systems biology approach to study innate and adaptive responses to vaccination against influenza in humans during three consecutive influenza seasons. We studied healthy adults vaccinated with trivalent inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV). TIV induced higher antibody titers and more plasmablasts than LAIV did. In subjects vaccinated with TIV, early molecular signatures correlated with and could be used to accurately predict later antibody titers in two independent trials. Notably, expression of the kinase CaMKIV at day 3 was inversely correlated with later antibody titers. Vaccination of CaMKIV-deficient mice with TIV induced enhanced antigen-specific antibody titers, which demonstrated an unappreciated role for CaMKIV in the regulation of antibody responses. Thus, systems approaches can be used to predict immunogenicity and provide new mechanistic insights about vaccines.

• Multiple innate immune pathways contribute to the immunogenicity of recombinant adenovirus vaccine vectors.

  Authors: Elizabeth G Rhee, Joseph N Blattman, Sudhir P Kasturi, R Phelps Kelley, David R Kaufman, Diana M Lynch, Annalena La Porte, Nathaniel L Simmons, Sarah L Clark, Bali Pulendran, Philip D Greenberg, Dan H Barouch

  Journal of virology. 10/2010; 85(1):315-23.

  The innate immune pathways that contribute to the potent immunogenicity of recombinant adenovirus (rAd) vaccine vectors remain largely undefined. Previous studies assessing innate immunity triggeredThe innate immune pathways that contribute to the potent immunogenicity of recombinant adenovirus (rAd) vaccine vectors remain largely undefined. Previous studies assessing innate immunity triggered by vaccine vectors have largely focused on in vitro studies involving antigen-presenting cells and on early in vivo inflammatory responses. Here, we systematically explore the Toll-like receptor (TLR) signaling requirements for the generation of cellular immune responses by intramuscular immunization with common and alternative serotype rAd vectors in mice. Antigen-specific CD8(+) T-lymphocyte responses elicited by these rAd vectors were significantly diminished in MyD88(-/-) mice but not in TRIF(-/-) or TLR3(-/-) mice, suggesting the importance of MyD88-dependent TLR signaling. However, the absence of each individual TLR resulted in minimal to no effect on vaccine-elicited cellular immune responses. Moreover, responses were not diminished in IL-1R(-/-) or IL-18R(-/-) mice. These data suggest that rAd vectors engage multiple MyD88-dependent signaling pathways, none of which are individually critical; rather, they are integrated to contribute to the potent immunogenicity of rAd vectors. Stimulation of multiple innate immune mechanisms may prove a generalizable property of potent vaccines, and this strategy could be harnessed in the development of next-generation vaccine vectors and adjuvants.

• Systems vaccinology.

  Authors: Bali Pulendran, Shuzhao Li, Helder I Nakaya

  Immunity. 10/2010; 33(4):516-29.

  Vaccination is one of the greatest triumphs of modern medicine, yet we remain largely ignorant of the mechanisms by which successful vaccines stimulate protective immunity. Two recent advances areVaccination is one of the greatest triumphs of modern medicine, yet we remain largely ignorant of the mechanisms by which successful vaccines stimulate protective immunity. Two recent advances are beginning to illuminate such mechanisms: realization of the pivotal role of the innate immune system in sensing microbes and stimulating adaptive immunity, and advances in systems biology. Recent studies have used systems biology approaches to obtain a global picture of the immune responses to vaccination in humans. This has enabled the identification of early innate signatures that predict the immunogenicity of vaccines, and identification of potentially novel mechanisms of immune regulation. Here, we review these advances and critically examine the potential opportunities and challenges posed by systems biology in vaccine development.

• Activation of beta-catenin in dendritic cells regulates immunity versus tolerance in the intestine.

  Authors: Santhakumar Manicassamy, Boris Reizis, Rajesh Ravindran, Helder Nakaya, Rosa Maria Salazar-Gonzalez, Yi-Chong Wang, Bali Pulendran

  Science (New York, N.Y.). 08/2010; 329(5993):849-53.

  Dendritic cells (DCs) play a vital role in initiating robust immunity against pathogens as well as maintaining immunological tolerance to self antigens. However, the intracellular signaling networksDendritic cells (DCs) play a vital role in initiating robust immunity against pathogens as well as maintaining immunological tolerance to self antigens. However, the intracellular signaling networks that program DCs to become tolerogenic remain unknown. We report here that the Wnt-beta-catenin signaling in intestinal dendritic cells regulates the balance between inflammatory versus regulatory responses in the gut. beta-catenin in intestinal dendritic cells was required for the expression of anti-inflammatory mediators such as retinoic acid-metabolizing enzymes, interleukin-10, and transforming growth factor-beta, and the stimulation of regulatory T cell induction while suppressing inflammatory effector T cells. Furthermore, ablation of beta-catenin expression in DCs enhanced inflammatory responses and disease in a mouse model of inflammatory bowel disease. Thus, beta-catenin signaling programs DCs to a tolerogenic state, limiting the inflammatory response.

• Programming dendritic cells to induce T(H)2 and tolerogenic responses.

  Authors: Bali Pulendran, Hua Tang, Santhakumar Manicassamy

  Nature immunology. 08/2010; 11(8):647-55.

  A fundamental puzzle in immunology is how the immune system decides what types of immune responses to launch against different stimuli. Although much is known about control of T helper type 1 (T(H)1)A fundamental puzzle in immunology is how the immune system decides what types of immune responses to launch against different stimuli. Although much is known about control of T helper type 1 (T(H)1) and T(H)17 responses, the mechanisms that initiate T(H)2 and T regulatory (T(reg)) responses remain obscure. Emerging studies suggest a fundamental role for the innate immune system, particularly dendritic cells (DCs), in this process. We review these studies, and suggest that the innate control of T(H)2 and T(reg) responses can be viewed as different hierarchies of organization, in which DCs, their innate receptors and signaling networks, and their interactions with other cells and local microenvironments represent different levels of the hierarchy.

• CD8+ T cell responses following replication-defective adenovirus serotype 5 immunization are dependent on CD11c+ dendritic cells but show redundancy in their requirement of TLR and nucleotide-binding oligomerization domain-like receptor signaling.

  Authors: Ross W B Lindsay, Patricia A Darrah, Kylie M Quinn, Ulrike Wille-Reece, Lisa M Mattei, Akiko Iwasaki, Sudhir P Kasturi, Bali Pulendran, Jason G D Gall, A Gregory Spies, Robert A Seder

  Journal of immunology (Baltimore, Md. : 1950). 08/2010; 185(3):1513-21.

  Replication-defective adenovirus serotype 5 (rAd5) is the most potent recombinant vector for eliciting CD8 T cell responses in humans. In this study, the innate mechanisms that influence T cellReplication-defective adenovirus serotype 5 (rAd5) is the most potent recombinant vector for eliciting CD8 T cell responses in humans. In this study, the innate mechanisms that influence T cell responses following rAd5 immunization were assessed in mice. Using rAd5 expressing enhanced GFP (eGFP-rAd5), we show that rAd5 transfects CD11c(+) dendritic cells (DCs) in draining lymph nodes in vivo following s.c. or i.m. immunization. Among distinct DC subsets, eGFP expression was highest in CD11c(+)CD8(-)B220(-) with a lower frequency detected in CD11c(+)CD8(+)B220(-) and CD11c(+)B220(+) plasmacytoid DCs. CD11c(+) DCs but not CD11c(-) cells from mice immunized with rAd5 encoding the SIINFEKL peptide induced proliferation of naive OT-I CD8 T cells. Furthermore, CD11c(+)CD8(+)B220(-) was the most potent DC subset for eliciting naive OT-I CD8 T cell proliferation. Of note, mice with pre-existing immunity to rAd5 had a substantial decrease in eGFP expression in DCs, which was associated with approximately 2-fold decrease in Th1 and complete inhibition of CD8 responses. Thus, pre-existing rAd5 immunity has a greater influence on CD8 compared with CD4 T cell responses. In terms of how innate cytokines and signaling pathways influenced T cell immunity following rAd5 immunization, we show that the magnitude and quality of CD8 T cell responses are partially dependent on MyD88 but independent of IL-12, type I IFN, apoptosis-associated speck-like protein, nucleotide-binding oligomerization domain-like receptor protein 3, and IL-1. Taken together, these data demonstrate a critical role for CD11c(+) DCs for CD8 responses but striking redundancy for innate cytokines and signaling by TLR and nucleotide-binding oligomerization domain-like receptor pathways.

• The T helper type 2 response to cysteine proteases requires dendritic cell-basophil cooperation via ROS-mediated signaling.

  Authors: Hua Tang, Weiping Cao, Sudhir Pai Kasturi, Rajesh Ravindran, Helder I Nakaya, Kousik Kundu, Niren Murthy, Thomas B Kepler, Bernard Malissen, Bali Pulendran

  Nature immunology. 07/2010; 11(7):608-17.

  The mechanisms that initiate T helper type 2 (T(H)2) responses are poorly understood. Here we demonstrate that cysteine protease-induced T(H)2 responses occur via 'cooperation' between migratoryThe mechanisms that initiate T helper type 2 (T(H)2) responses are poorly understood. Here we demonstrate that cysteine protease-induced T(H)2 responses occur via 'cooperation' between migratory dermal dendritic cells (DCs) and basophils positive for interleukin 4 (IL-4). Subcutaneous immunization with papain plus antigen induced reactive oxygen species (ROS) in lymph node DCs and in dermal DCs and epithelial cells of the skin. ROS orchestrated T(H)2 responses by inducing oxidized lipids that triggered the induction of thymic stromal lymphopoietin (TSLP) by epithelial cells mediated by Toll-like receptor 4 (TLR4) and the adaptor protein TRIF; by suppressing production of the T(H)1-inducing molecules IL-12 and CD70 in lymph node DCs; and by inducing the DC-derived chemokine CCL7, which mediated recruitment of IL-4(+) basophils to the lymph node. Thus, the T(H)2 response to cysteine proteases requires DC-basophil cooperation via ROS-mediated signaling.

• Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus.

  Authors: Balaji Manicassamy, Santhakumar Manicassamy, Alan Belicha-Villanueva, Giuseppe Pisanelli, Bali Pulendran, Adolfo García-Sastre

  Proceedings of the National Academy of Sciences of the United States of America. 06/2010; 107(25):11531-6.

  Influenza A virus is being extensively studied because of its major impact on human and animal health. However, the dynamics of influenza virus infection and the cell types infected in vivo areInfluenza A virus is being extensively studied because of its major impact on human and animal health. However, the dynamics of influenza virus infection and the cell types infected in vivo are poorly understood. These characteristics are challenging to determine, partly because there is no efficient replication-competent virus expressing an easily traceable reporter gene. Here, we report the generation of a recombinant influenza virus carrying a GFP reporter gene in the NS segment (NS1-GFP virus). Although attenuated when compared with wild-type virus, the NS1-GFP virus replicates efficiently in murine lungs and shows pathogenicity in mice. Using whole-organ imaging and flow cytometry, we have tracked the dynamics of influenza virus infection progression in mice. Imaging of murine lungs shows that infection starts in the respiratory tract in areas close to large conducting airways and later spreads to deeper sections of the lungs. In addition to epithelial cells, we found GFP-positive antigen-presenting cells, such as CD11b(+)CD11c(-), CD11b(-)CD11c(+), and CD11b(+)CD11c(+), as early as 24 h after intranasal infection. In addition, a significant proportion of NK and B cells were GFP positive, suggesting active infection of these cells. We next tested the effects of the influenza virus inhibitors oseltamivir and amantadine on the kinetics of in vivo infection progression. Treatment with oseltamivir dramatically reduced influenza infection in all cell types, whereas, surprisingly, amantadine treatment more efficiently blocked infection in B and NK cells. Our results demonstrate high levels of immune cells harboring influenza virus antigen during viral infection and cell-type-specific effects upon treatment with antiviral agents, opening additional avenues of research in the influenza virus field.