Zhengguo Xiao

Signals required for programming effector and memory development by CD8+ T cells

Summary:  Stimulation of naïve CD8+ T cells with antigen and costimulation results in proliferation and weak clonal expansion, but the cells fail to develop effector functions and are tolerant long term. Initiation of the program leading to the strong expansion and development of effector functions and memory requires a third signal that can be provided by interleukin‐12 (IL‐12) or interferon‐α (IFN‐α). CD4+ T cells condition dendritic cells (DCs) to effectively present antigen to CD8+ T cells, and this conditioning involves, at least in part, CD40‐dependent upregulation of the production of these signal 3 cytokines by the DCs. Upon being fully activated, the cytotoxic T lymphocytes develop activation‐induced non‐responsiveness (AINR), a form of split anergy characterized by an inability to produce IL‐2 to support continued expansion. If antigen remains present, IL‐2 provided by CD4+ T cells can reverse AINR to allow further expansion of the effector population and conversion to responsive memory cells following antigen clearance. If IL‐2 or potentially other proliferative signals are not available, persistent antigen holds cells in the AINR state and prevents the development of a responsive memory population. Thus, in addition to antigen and costimulation, CD8+ T cells require cytokine signals at distinct stages of the response to be programmed for optimal generation of effector and memory populations.

Immunological Responses of Swine to Porcine Reproductive and Respiratory Syndrome Virus Infection

The immunology of porcine reproductive and respiratory syndrome virus (PRRS) begins with an initial encounter of PRRSV with the pig. Regardless of the route of entry of PRRSV--via inhalation, intramuscular vaccination, insemination, or other routes--productive infection occurs predominately in alveolar macrophages of the lung. Thus, innate responses of the lung and the alveolar macrophage comprise the initial defense against PRRSV. The virus appears not to elicit innate interferon and cytokine responses characteristic of other strongly immunogenic viral pathogens, and its effects are consistent with induction of a weak adaptive immune response. Humoral and cell-mediated immunity is induced in due course, and results in clearance of virus from the circulation but not from lymphoid tissues, where the infection becomes persistent. Subsequent reexposure to PRRSV elicits an anamnestic response that is partially to completely protective. Within this unconventional picture of anti-PRRSV immunity lie a variety of unresolved issues, including the nature of protective immunity within individual pigs and among pigs in commercial populations, the efficacy of protective immunity against genetically different PRRSV isolates, the effects of developmental age, sex, genetics, and other host factors on the immune response to PRRSV, and the possible suppression of host immunity to other pathogens.

Programming for CD8 T cell memory development requires IL-12 or type I IFN.

Inflammation can have both positive and negative effects on development of CD8 T cell memory, but the relative contributions and cellular targets of the cytokines involved are unclear. Using CD8 T cells lacking receptors for IL-12, type I IFN, or both, we show that these cytokines act directly on CD8 T cells to support memory formation in response to vaccinia virus and Listeria monocytogenes infections. Development of memory to vaccinia is supported predominantly by IL-12, whereas both IL-12 and type I IFN contribute to memory formation in response to Listeria. In contrast to memory formation, the inability to respond to IL-12 or type I IFN had a relatively small impact on the level of primary expansion, with at most a 3-fold reduction in the case of responses to Listeria. We further show that programming for memory development by IL-12 is complete within 3 days of the initial naive CD8 T cell response to Ag. This programming does not result in formation of a population that expresses killer cell lectin-like receptor G1, and the majority of the resulting memory cells have a CD62Lhigh phenotype characteristic of central memory cells. Consistent with this, the cells undergo strong expansion upon rechallenge and provide protective immunity. These data demonstrate that IL-12 and type I IFN play an essential early role in determining whether Ag encounter by naive CD8 T cells results in formation of a protective memory population.

The Level of Virus-Specific T-Cell and Macrophage Recruitment in Porcine Reproductive and Respiratory Syndrome Virus Infection in Pigs Is Independent of Virus Load

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important infectious disease agent of pigs worldwide, causing reproductive failure in pregnant sows and respiratory problems in nursing and growing pigs. PRRSV infection is characterized by a prolonged viremia of 30 or more days and an extended persistent infection of lymphoid tissues. To better understand the immunological basis for prolonged acute and persistent PRRSV infection, we have examined the cell-mediated immune (CMI) response throughout the course of infection and compared the results to the local distribution and abundance of PRRSV in infected tissues. PRRSV-specific T cells, enumerated by gamma interferon enzyme-linked immunospot assay, did not appear until 2 weeks after PRRSV inoculation, and their abundance exhibited substantial variation over time and among animals. In all cases the T-cell response was transient. High levels of viral RNA were present in lymphoid tissues of all animals in the acute phase of infection. Viral loads were decreased 1,000-fold or more in persistent infections, with the primary sites of persistence being tonsil, sternal lymph node, and inguinal lymph node. The abundance of virus-specific T cells in either acutely or persistently infected animals was highly variable and showed no correlation to the level of virus in lymphoid tissues. No significant difference in antigen-specific T-cell abundance was observed in secondary lymphoid tissues in either acute or persistent infection except for tonsil, in which the number of responding cells was extremely low. CD4+- and CD8+-T-cell frequencies did not change after PRRSV infection, though a decrease in γδ T cells was observed. Macrophages, the permissive cell type for PRRSV, were present in various levels in all tissue preparations and were not in proportion to local virus load. These findings indicate that a weak CMI response contributes to prolonged PRRSV infection and suggests that PRRSV suppresses T-cell recognition of infected macrophages. Thus, the slow but eventual resolution of PRRSV infection may be dependent on limiting permissive macrophages and on innate immune factors.

Detuning CD8 T cells: down-regulation of CD8 expression, tetramer binding, and response during CTL activation

CD8 is critical for T cell recognition of peptide/class I major histocompatability complex ligands, yet is down-regulated during activation of CD8 T cells. We report that loss of CD8 expression early during in vivo responses to vaccinia virus or Listeria monocytogenes (LM) correlates with decreased T cell staining with specific class I/peptide tetramers and reduced CD8 T cell sensitivity for antigen. Loss of CD8 cell surface expression occurs despite sustained mRNA expression, and CD8 levels return to normal levels during differentiation of memory cells, indicating a transient effect. We determined that during response to LM, CD8 down-regulation is regulated by T cell reactivity to type I interferon (IFN-I) because CD8 loss was averted on IFN-I receptor–deficient T cells. IFN-I alone was not sufficient to drive CD8 down-regulation, however, as antigen was also required for CD8 loss. These results suggest that CD8 effector T cell differentiation involves a transient down-regulation of antigen sensitivity (CTL “detuning”), via reduced CD8 expression, a feature that may focus the effector response on target cells expressing high levels of antigen (e.g., infected cells), while limiting collateral damage to bystander cells.

Species specialization in cytokine biology: Is interleukin-4 central to the TH1–TH2 paradigm in swine?

The TH1-TH2 paradigm provides an elegant model of directed response to infectious pathogens. Developed in the mouse, the model has provided a framework for systematic and mechanistic studies of immune regulation, protective immunity, and vaccine development in swine. Interleukin-4 (IL-4) plays a central role in the paradigm as a regulatory molecule directing development of the TH2 phenotype, as a developmental cytokine essential for antibody production, and as a soluble diagnostic marker of the TH2 cell type. In contrast, while characterizing the biological properties of porcine IL-4, we discovered that it was not a stimulatory factor for porcine B cells. Rather, it blocked antibody and IL-6 secretion and suppressed antigen-stimulated proliferation of B cells. Inhibition was not reversed by treatment with IL-2 and IL-6 treatment. IL-4 did not stimulate T lymphocyte proliferation, but induced cell growth in lymphoblasts in a dose-dependent fashion. These results suggest that IL-4 plays a different role in pigs than in mice and humans, in which it stimulates B cells and is essential for antibody production. Furthermore, the functions of IL-4 in swine cannot be inferred from results in model systems such as the mouse. General models of disease resistance show substantial variation between pigs and mice at the cellular and molecular level. Advances in somatic cell technologies and animal engineering to enable gene knockouts in pigs, in combination with a continuously expanding immunological toolkit, promise an exciting future for pig immunology, detailed mechanistic elucidation of the TH1-TH2 paradigm, and an improved understanding of the role of IL-4 in porcine immunity to infectious disease.

Cholera toxin activates nonconventional adjuvant pathways that induce protective CD8 T-cell responses after epicutaneous vaccination

The ability to induce humoral and cellular immunity via antigen delivery through the unbroken skin (epicutaneous immunization, EPI) has immediate relevance for vaccine development. However, it is unclear which adjuvants induce protective memory CD8 T-cell responses by this route, and the molecular and cellular requirements for priming through intact skin are not defined. We report that cholera toxin (CT) is superior to other adjuvants in its ability to prime memory CD8 T cells that control bacterial and viral challenges. Epicutaneous immunization with CT does not require engagement of classic toll-like receptor (TLR) and inflammasome pathways and, surprisingly, is independent of skin langerin-expressing cells (including Langerhans cells). However, CT adjuvanticity required type-I IFN sensitivity, participation of a Batf3-dependent dendritic cell (DC) population and engagement of CT with suitable gangliosides. Chemoenzymatic generation of CT–antigen fusion proteins led to efficient priming of the CD8 T-cell responses, paving the way for development of this immunization strategy as a therapeutic option.

TLR agonists are highly effective at eliciting functional memory CTLs of effector memory phenotype in peptide immunization.

Given the importance of memory cytotoxic T lymphocytes (CTLs) in eliminating altered self-cells, including virus-infected and tumor cells, devising effective vaccination strategies for generating memory CTLs is a priority in the field of immunology. Herein, we elaborate upon a novel boosting approach that utilizes synthetic peptides and Toll-like receptor (TLR) agonists as adjuvants to generate sufficient numbers of memory CTLs to protect against infection in mice. Peptide boosting with lipopolysaccharide (LPS), a TLR4-ligand, has been shown to progressively enhance memory CTLs. Whether this result is strictly dependent on activation of TLR4 or can be similarly achieved by signaling through other TLRs is of practical interest in vaccine development but is yet unknown. In this report, we present evidence that intravenous peptide boosting together with TLR3 and TLR9 agonists (Poly IC and CpG, respectively) is highly effective and induces large quantities of memory CTLs of effector memory phenotype after three boosts. Compared to LPS, CpG and Poly IC generate more robust immune responses after the first and second boosts, indicating that a protective level of CTLs might be achieved with fewer boosts when CpG or Poly IC is used. Lastly, the resultant memory CTLs from boosting with different TLR agonists as adjuvant are equally protective against pathogen challenge and are not immune senescent. Therefore, TLR agonists are effective adjuvants in intravenous peptide boosting for the generation of functional memory CTLs.

Temporal regulation of rapamycin on memory CTL programming by IL-12.

Mammalian target of rapamycin (mTOR) is a master regulator of cell growth. Recent reports have defined its important role in memory cytotoxic T lymphocyte (CTL) differentiation in infections and memory programming. We report that rapamycin regulated memory CTL programming by IL-12 to a similar level in a wide range of concentrations, and the enhanced memory CTLs by rapamycin were functional and provided similar protection against Listeria Monocytogenes challenge compared to the control. In addition, rapamycin-experienced CTLs went through substantially enhanced proliferation after transfer into recipients. Furthermore, the regulatory function of rapamycin on CD62L expression in memory CTLs was mainly contributed by the presence of rapamycin in the first 24-hr of stimulation in vitro, whereas the effective window of rapamycin on the size of memory CTLs was determined between 24 to 72 hrs. In conclusion, rapamycin regulates IL-12-driven programming of CTLs to a similar level in a wide range of concentrations, and regulates the phenotype and the size of memory CTLs in different temporal windows.

Detection of porcine reproductive and respiratory syndrome virus in pigs with low positive or negative ELISA S/p ratios

DipACVM, M. Olin, BS, T. W. Molitor, MS, PhD, H. S. Joo, DVM, PhD, C. Pijoan, DVM, PhD, Swine Disease Eradication Center, Room 385C, University of Minnesota College of Veterinary Medicine, 1988 Fitch Avenue, St Paul, MN 55108, USA K. D. Rossow, DVM, PhD, Minnesota Veterinary Diagnostic Laboratory, 1333 Gortner Avenue, St Paul, MN 55108, USA D. D. Polson, DVM, MS, PhD, Boeheringer Ingelheim Vetmedica 2501, North Loop Drive 1000, Ames, IA 50010, USA Z. Xiao, DVM, M. P. Murtaugh, PhD, Department of Veterinary Pathobiology, University of Minnesota College of Veterinary Medicine, 1971 Commonwealth Avenue, St Paul, MN 55108, USA