A comprehensive method for the phenotypical and functional characterization of recalled human memory B and T cells specific to vaccine antigens.

Current methodologies for assessing vaccine effectiveness and longevity primarily center on measuring vaccine-induced neutralizing antibodies in serum or plasma. However, these methods overlook additional parameters such as the presence of memory B cells, even as antibody levels wane, and the pivotal role played by memory T cells in shaping antigen-specific memory B cell responses. Several studies have employed a combination of polyclonal activators, such as CpG and R848, along with various cytokines to provoke the recall of memory B cells from peripheral blood mononuclear cells (PBMCs) into antibody-secreting cells (ASCs). Other studies have examined the use of live attenuated viruses to stimulate antigen-specific memory T cells within PBMCs into effector T cells that produce Th1/Th2 cytokines. However, these studies have not fully elucidated the distinct effects of these polyclonal activators on individual subsets, nor have they evaluated whether the vaccine antigen alone is sufficient to trigger the recall of memory T cells. Thus, in this study, we directly compared the capacity of two B cell polyclonal activators to induce the transition of existing vaccine-specific memory cells present in peripheral blood samples into ASCs. Simultaneously, we also assessed the transition of existing memory T cells into effector subsets in response to vaccine antigens. Our findings demonstrate that both polyclonal activator combinations, CpG with IL-6 and IL-15, as well as R848 with IL-2, effectively induce the terminal differentiation of memory B cells into ASCs. Notably, CpG treatment preferentially expanded naïve and non-class-switched B cells, while R848 expanded class-switched memory cells, plasmablasts, and plasma cells. Consequently, R848 treatment led to a greater overall production of total and antigen-specific IgG immunoglobulins. Additionally, the exposure of isolated PBMCs to vaccine antigens alone proved sufficient for recalling the rare antigen-specific memory T cells into effector subsets, predominantly consisting of IFN-γ-producing CD4 T cells and TNF-ß-producing CD8 T cells. This study not only establishes a rationale for the selection of methods to expand and detect antigen-specific lymphocyte subsets but also presents a means to quantify vaccine effectiveness by correlating serum antibody levels with preexisting memory cells within peripheral blood samples.

Human T cells show plasticity for direct recognition of xenogeneic dendritic cells.

Organ shortage continues to be the forefront of problems facing clinical transplantation. Although xenografts serve as a promising alternative, its success is contingent upon further investigation into the mechanisms of cell-mediated xenograft rejection. Here, we explored the direct and indirect contribution of human immune cells in xenorecognition using human and murine in vitro coculture systems. Our data shows that human T cells directly recognized the xenogeneic MHC molecules since blocking of MHCs suppressed their proliferative response and cytokines production of IL-2 and IFN-γ. While B and NK cells alone did not generate a significant response, the combination of B and T cells promoted indirect xenorecognition by T cells as evidenced by an increase in B cell proliferative response. Overall, our data suggests that human T cells have the plasticity to recognize xenogeneic MHCs and contribute to xenograft rejection.

TLR3 and TLR7/8 agonists improve immunization outcome in nicotine exposed mice through different mechanisms.

It is known that cigarette smoke compromises the immune system and increases the risk of vaccine-preventable diseases. We reported that nicotine, the immunosuppressive component of cigarette smoke, disrupts the functional properties of DC and DC crosstalk with NK cells, which is pivotal in the initiation of immune responses to vaccines. We also showed that select TLR agonists could reduce the degrading effects of nicotine on DC-NK mediated immune responses in vitro. In this study, we further investigated the magnitude and mechanism of immune responses to a protein antigen formulated with alum or TLR agonists using WT, NK-depleted, and IFN-γ deficient mice exposed to nicotine. We found that R848 followed by Poly(I:C) acted as the most effective adjuvants to increase the percentage and number of IFN-γ-producing effector NK cells in the lymph nodes of immunized mice. In addition, we observed that the protein antigen formulated with Poly(I:C) or R848 improved the antigen-specific immune response in nicotine-exposed mice through NK-independent and -dependent mechanisms, respectively. These findings extend our understanding of the hyporesponsiveness of smokers to vaccines and provides the means to increase the efficacy of vaccines in this large population.

R848/TLR7-Mediated Stronger CD8+ T Immunity Is Dependent on DC-NK Cell Interactions.

BACKGROUND: Toll-like receptor (TLR) 7 agonists are effective candidates for Th1 immune adjuvants, which compensate for the insufficient Th1 immune responses induced by traditional adjuvants. This effect is currently dependent on TLR7-mediated induction of dendritic cell (DC) maturation and increased IL-12 production. METHODS: In vivo, we intraperitoneally injected TLR agonists with OVA, and LNs were collected for detection. In vitro, Activated DCs, natural killer (NK) cells, and CD8+ T cells were tested using flow cytometry for surface expression and enzyme-linked immunosorbent assay for cytokine production. NK cell migration was evaluated using transwell system. All experiments were performed in both C57BL/6 and BALB/C backgrounds. RESULTS: Our findings revealed that the enhanced CD8+ T immunity characterized by CD8+ T accumulation, proliferation, and IFN-γ+CD8+ T induction induced by R848 was attributed to DC-dependent NK cell migration and DC-NK interactions. Our results demonstrated that R848 induced CD8+ T cell accumulation and IFN-γ+CD8+ T cells in lymph nodes (LNs) to a greater degree in vivo than TLR4 agonists (lipopolysaccharide) and TLR9 agonists (Class C CPG). R848-activated DCs enhanced CD8+ T cell proliferation and increased IFN-γ+CD8+ T cells with the assistance of NK cells. In contrast, depletion of NK cell decreased IFN-γ+CD8+ T cell production. Greater NK cell migration to LNs occurred in R848-immunized mice. A similar effect of R848 on NK cell migration was observed in an in vitro transwell study. When co-cultured, NK cells plus R848 could promote DCs maturation, and in turn, DCs in combination of R848 augmented NK cells activation. Further studies demonstrated that among several TLR agonists, R848 produced the largest amount of the chemokine CXCL9 from activated DCs, which is relevant to NK cell migration. CXCL9 blockade reduced the number of migrated NK cells, and the addition of CXCL9 increased the number of NK cells. DISCUSSION: Taken together, R848-mediated stronger CD8+ T cell immunity does not depend on DC activation alone, rather that NK cells must also be considered. By increasing our immunological understanding of the effect of R848/TLR7, these findings provide a new perspective for applying R848 in future clinical studies.

Combination of TLR8 and TLR4 agonists reduces the degrading effects of nicotine on DC-NK mediated effector T cell generation.

The magnitude of immune responses to vaccination is a critical factor in determining protection from disease. It is known that cigarette smoke dampens the immune system and increases the risk of vaccine-preventable diseases. We reported that nicotine, the immunosuppressive component of cigarette smoke, disrupts the differentiation and functional properties of DC, which are pivotal in the initiation of immune response to vaccines. We also reported that TLR agonists act in synergy and boost DC maturation, DC-NK crosstalk and ultimately naïve T cell polarization into effector Th1 and Tc1 cells. Here, we investigated whether the combination of TLR agonists could diminish the degrading effects of nicotine on DC-NK mediated effector T cell generation. We found that none of TLR agonists, single or combined, were able to diminish completely the adverse effects of nicotine on DC. However, TLR3, TLR4, and TLR8 agonists acted as the most effective adjuvants to increase the expression levels of antigen-presenting, costimulatory molecules and production of cytokines by nicotine-exposed DC (nicDC). When combined, TLR3 + 8 and TLR4 + 8 synergistically optimized nicDC maturation and IFN-γ secretion from nicotine-exposed NK (nicNK) during co-cultures. Interestingly, in contrast to DC-NK-T, co-cultures of nicDC-nicNK-T treated with TLR3 + 8 or TLR4 + 8 agonists produced a similar frequency of effector memory Th1 and Tc1 cells. However, the effector cells from TLR4 + 8 followed by TLR3 + 8 treated nicDC-nicNK-T co-cultures produced significantly more IFN-γ when compared with aluminum salt treated co-culture. Our data suggest that addition of appropriate TLR agonists to vaccine formulation could potentially augment the immune response to vaccination in smokers.

TLR8 combined withTLR3 or TLR4 agonists enhances DC-NK driven effector Tc1 cells.

BACKGROUND: Most current prophylactic vaccines confer protection primarily through humoral immunity. Indeed, aluminum salts which have been widely used as adjuvants in vaccines primarily enhance Th2-driven antibody responses. Therefore, new vaccines formulation is moving toward a careful selection of adjuvants that also elicit significant Th1 or Tc1 responses. Several TLR agonists have been tested as potential new adjuvants in clinical and preclinical studies with some efficacy. These studies suggest that combining more than one of TLR ligands enhances the magnitude of immune responses to cancer and infectious disease. OBJECTIVES: In order to evaluate the synergistic effect of TLR agonists for effective induction of cellular immunity, we investigated the effects of single and/or combined TLR agonists on monocyte-derived DC maturation, DC-NK crosstalk and ultimately naïve T cells polarization into effector T cells. RESULTS: Among the adjuvants tested, we found that TLR3, TLR4, TLR7/8 and TLR8 agonists were the most effective adjuvants to increase the expression levels of antigen-presenting, co-stimulatory molecules and production of cytokines by maturing DCs. When combined, TLR3+8 and TLR4+8 synergistically optimized DC maturation and IFN-γ secretion from NK cells co-cultured with DCs. Interestingly, co-culture of DC-NK-T treated with aluminum salt produced the highest percentage of effector memory CFSE-CCR7- Th1 cells whereas TLR3+8 and TLR4+8 treated co-cultures produced the highest percentage of effector memory CFSE-CCR7- Tc1 cells producing IFN-γ. Finally, while both TLR3+8 or TLR4+8 treated co-cultures generated similar frequency of Th1 and Tc1 effector cells, the effector cells from the latter co-culture produced quantitatively more IFN-γ in the supernatant. CONCLUSION: Our data indicate that if in need of an enhanced DC-NK mediated cellular immunity one may select TLR agonists with defined synergistic effects.

Phenotyping and comparing the immune cell populations of free-ranging Atlantic bottlenose dolphins (Tursiops truncatus) and dolphins under human care.

BACKGROUND: Studies suggest that free-ranging bottlenose dolphins exhibit a suppressed immune system because of exposure to contaminants or microorganisms. However, due to a lack of commercially available antibodies specific to marine mammal immune cell surface markers, the research has been indecisive. The purpose of this study was to identify cross-reactive terrestrial-specific antibodies in order to assess the changes in the immune cell populations of dolphins under human care and free-ranging dolphins. The blood and PBMC fraction of blood samples from human care and free-ranging dolphins were characterized by H&E staining of cytospin slides and flow cytometry using a panel of terrestrial-specific antibodies. RESULTS: In this study, we show that out of 65 terrestrial-specific antibodies tested, 11 were cross-reactive and identified dolphin immune cell populations within their peripheral blood. Using these antibodies, we found significant differences in the absolute number of cells expressing specific markers within their lymphocyte and monocyte fractions. Interestingly, the peripheral blood mononuclear cell profile of free-ranging dolphins retained an additional population of cells that divided them into two groups showing a low (<27%) or high (>56%) percentage of smaller cells resembling granulocytes. CONCLUSIONS: We found that the cross-reactive antibodies not only identified specific changes in the immune cells of free-ranging dolphins, but also opened the possibility to investigate the causal relationship between immunosuppression and mortality seen in free-ranging dolphins.

Exposure to nicotine adversely affects the dendritic cell system and compromises host response to vaccination.

The magnitude of Th1 cells response to vaccination is a critical factor in determining protection from clinical disease. Our previous in vitro studies suggested that exposure to the nicotine component of cigarette smoke skews the differentiation of both human and mouse dendritic cell (DC) precursors into atypical DCs (DCs differentiated ex vivo in the presence of nicotine) lacking parameters essential for the development of Th1-mediated immunity. In this study, we determined the causal relationship between nicotine-induced DC alterations and host response to vaccines. We show that animals exposed to nicotine failed to develop and maintain Ag-specific effector memory Th1 cells and Ab production to protein-based vaccine formulated with Th1 adjuvants. Accordingly, both prophylactic and therapeutic vaccines failed to protect and cure the nicotine-exposed mice from disease. More importantly, we demonstrate the nicotine-induced defects in the biological activities of in vivo DCs as an underlying mechanism. Indeed, i.v. administration of DCs differentiated in the presence of nicotine preferentially promoted the development of Ag-specific IL-4-producing effector cells in the challenged mice. In addition, DC subsets isolated from mice exposed to nicotine produced significantly less cytokines in response to Th1 adjuvants and inadequately supported the development of Ag-specific Th1 cells. Collectively, our studies suggest that nicotine-induced defects in the DC system compromises vaccine efficacy in smokers.

Chitin particles induce size-dependent but carbohydrate-independent innate eosinophilia.

Murine Mϕ that phagocytose CMP develop into M1; this response depends on the size and the chemical composition of the particles. In contrast, recent studies concluded that chitin particles induce M2 and eosinophil migration, promoting acquired Th2 immune responses against chitin-containing microbes or allergens. This study examined whether these apparently inconsistent responses to chitin could be induced by variation in the size and chemical composition of the chitin particles. We compared the responses of Mϕ with CMP, LCB, and Sephadex G-100 beads (>40 µm). Beads were given i.p. to WT mice and to mice deficient in a CRTH2, a receptor for the eosinophil chemoattractant PGD(2). In contrast to the M1 activation induced by CMP, i.p. administration of LCB or Sephadex beads induced within 24 h a CRTH2-dependent peritoneal eosinophilia, as well as CRTH2-independent activation of peritoneal Mϕ that expressed Arg I, an M2 phenotype. LCB-induced Mϕ exhibited elevated Arg I and a surface MR, reduced surface TLR2 levels, and no change in the levels of CHI3L1 or IL-10 production. Our results indicate that the effects of chitin in vivo are highly dependent on particle size and that large, nonphagocytosable beads, independent of their chemical composition, induce innate eosinophilia and activate Mϕ expressing several M2, but not M1, phenotypes.

Comparative analysis of dendritic cells and anti-CD3/CD28 expanded regulatory T cells for application in transplantation.

The containment of direct and indirect recognition of donor alloantigens by enhancement of the number and activity of regulatory T cells (Tregs) has been one of the promising approaches to achieve transplant tolerance. Two major methods, dendritic cell (DC) and anti-CD3/CD28 antibodies (Abs) have been introduced for ex vivo expansion of Tregs prior to their adoptive transfer. Here we compared the clinical advantage of using these methods of Tregs expansion by evaluating the nature and ability of expanded Tregs to abolish recipient alloreactive T-cell responses in vitro and in vivo. The ovalbumin (OVA)-specific Tregs isolated from DO11.10 mice were exposed to either Abs or syngeneic DC loaded with OVA peptide in the presence of exogenous IL-2 for a week. Using BABL/c as recipient and C57BL/6 as donor, the suppressive activity of these cells was examined. We found that DCs were much more efficient than Abs in expanding (9-fold versus 4-fold) and maintaining the viability (90% versus 35%) of purified Tregs. Interestingly, the Abs-expanded Tregs superbly contained the alloreactive T-cell proliferation and both Tregs were more suppressive when the Tregs cognate antigen and alloantigens were separately expressed on recipient and donor DCs (HVGD) rather than on recipient DC alone (GVHD). Importantly, however, DC-expanded Tregs maintained stable expression of Foxp3, survived longer and effectively contained the differentiation of alloreactive T cells into IFN-gamma-producing effector cells both in vitro and in vivo. Our data suggests that DC-expanded Tregs provides a clinically advantageous means of preventing unwanted immune reactions to allografts.

B7-1 and B7-2 differentially control peripheral homeostasis of CD4(+)CD25(+)Foxp3(+) regulatory T cells.

Targeting the CD28/B7 interaction remains among the most promising approaches to treat transplant rejection. A drawback to this approach is however the observations of decreased numbers of naturally occurring regulatory T cells (Tregs) in CD28(-) or B7-deficient non-obese diabetic (NOD) mice, cells that maintain immunological self tolerance, prevent autoimmunity and control immune responses to transplants. In this study, therefore, we investigated the relative contributions of B7-1 and B7-2, the only known ligands of CD28, on the thymic development and peripheral homeostasis of Tregs. Our data indicates that the absence of both B7-1 and B7-2 result in a dramatic reduction in the frequencies of Tregs in thymus and peripheral tissues of B7-1/B7-2-deficient mice with no apparent changes in the percentage and distribution of conventional T-cell subsets. In addition, neither B7-1 nor B7-2 expression alone is sufficient for the development and peripheral homeostasis of Tregs. Interestingly, while B7-1 and B7-2 equally contribute to thymic development of Tregs, the significant loss in peripheral homeostasis of Tregs is more evident in the absence of B7-2 as compared to B7-1. Consistent with these results we found that B7-2-deficient DCs are less effective than B7-1-deficient DCs in maintaining Tregs in vitro due to their inability to induce IL-2 production by conventional T cells and sustain Tregs expression of CD25. This study provides the first demonstration of relative roles of B7-1 and B7-2 in Tregs homeostasis and suggests that therapeutic approaches designed to selectively interrupt CD28/B7-2 interaction could indeed have measurable impact on sustaining Tregs homeostasis.

Dendritic cells in transplant tolerance.

Dendritic cells (DCs) are a heterogeneous family of professional APCs involved in priming adaptive immune responses. Donor DCs (direct pathway of allorecognition) and recipient DCs presenting processed donor major histocompatibility complex (MHC) as peptides (indirect pathway of allorecognition) participate actively in graft rejection by stimulating recipient T cell responses following organ transplantation. Recent studies have shown that DCs also play a central role in inducing and maintaining tolerance to self antigens (Ags) through deletion, anergy, and regulation mechanisms. It is easy to see how the remarkable functional plasticity of DCs renders them attractive therapeutic targets for immune modulation. Indeed, in the past few years, successful outcomes in rodent models have built the case that DC-based therapy may provide a novel approach to transplant tolerance. Ongoing research into our understanding of the mechanisms whereby DCs promote tolerance in the steady-state, together with development of biologically, pharmacologically and genetically manipulated ex vivo DCs to mimic/enhance their natural tolerogenicity, should warrant the success of these experimental DCs in establishing long-term allograft survival.

Dendritic cells as promoters of transplant tolerance.

Dendritic cells (DCs) play a crucial role during the initiation of immune responses against non-self antigens. Following organ transplantation, activated donor- and recipient-derived DCs participate actively in graft rejection by sensitising recipient T cells via the direct or indirect pathways of allorecognition, respectively. There is increasing evidence that immature/semi-mature DCs induce antigen-specific unresponsiveness or tolerance to self antigens, both in central lymphoid tissue and in the periphery, through a variety of mechanisms (deletion, anergy and regulation). In the past few years, DC-based therapy of experimental allograft rejection has focused on ex vivo biological, pharmacological and genetic engineering of DCs to mimic/enhance their natural tolerogenicity. Successful outcomes in rodent models have built the case that DC-based therapy may provide a novel approach to transplant tolerance. Ongoing research into the role that DCs play in the induction of tolerance should allow for its clinical application in the near future.

A possible mechanism linking cigarette smoke to higher incidence of respiratory infection and asthma.

T helper type 1 (Th1) cells are responsible for cell-mediated immunity against invading pathogens, while Th2 cells provide help to B cells and control allergic responses. The polarization of naïve Th cells into Th1 or Th2 subsets is controlled by dendritic cells (DCs) migrating from the periphery to draining lymph nodes. Migrating DCs carry not only antigen-specific 'signal 1' and costimulatory 'signal 2', but also Th-polarizing 'signal 3' that reflects the nature of the pathogen and the character of the infected tissue. Any changes imposed by external factors on the DC lifecycle may result in an inappropriate immune response. Here we show that DCs developed in a nicotinic environment (nicDCs) fail to support the terminal development of effector memory Th1 cells due to their differential expression of costimulatory molecules and lack of IL-12 production. Interestingly, they adopt critical Th1-promoting function necessary to prevent and fight infections only when the total balance of environmental signals strongly favors Th1 immunity. Notably, in a Th2-biased environment, nicDCs provoke stronger than normal Th2 responses which predisposes the development and exacerbation of asthma. These results help explain the two opposing effects of cigarette smoke on respiratory immune defense mechanisms: (a) immunosuppression against infectious agents and (b) exacerbation of asthma.

Nicotinic environment affects the differentiation and functional maturation of monocytes derived dendritic cells (DCs).

Differentiation of tissue monocytes into DCs is a critical phase in the development of a competent immune system. We show that in a nicotinic environment, while human monocytes differentiate into DCs (henceforth called nicDCs) with a typical morphology, they display unique phenotype and cytokine profile that adversely affect their function. Despite an increased capacity for receptor-dependent antigen uptake, nicDCs do not express CD1a and fail to fully up-regulate MHCs, molecules essential for their antigen-presenting function. Additionally, in response to bacterial antigen LPS, maturing nicDCs hardly express the chemotactic cytokine receptor 7 required for their entry into lymphatic vessels. Furthermore, in parallel with their differential expression of costimulatory molecules CD80 and CD86 and lack of IL-12, nicDCs display profoundly reduced Th1 promoting capacity. These findings thus indicate that nicotine impedes the development of cell-mediated immunity by skewing DC differentiation. These effects of nicotinic environment on DC differentiation may contribute to the increased risks of respiratory tract infection and various cancers in smokers.

Evidence for the immunosuppressive role of nicotine on human dendritic cell functions.

Nicotine alters a wide range of immunological functions, including innate and adaptive immune responses. To date, no studies have been reported showing the immunoregulatory effects of nicotine on dendritic cells (DCs), which are critical cells for initiation of cell-mediated immunity against infection and neoplastic diseases. In this work, we report that, in a nicotinic environment, monocyte-derived DCs manifest lower endocytic and phagocytic activities. Interestingly, although immature DCs undergo maturation in response to bacterial antigen lipopolysaccharide, they produce decreased levels of pro-inflammatory cytokines, notably interleukin-12, and reveal a reduced ability to stimulate antigen-presenting cell-dependent T-cell responses. Importantly, the reduction in T-cell responses is associated with a diminished ability of DCs to induce differentiation and expansion of type 1 T cells, as evidenced by a decreased frequency of interferon-gamma-producing effector cells. These results strongly suggest that nicotine can exert its immunosuppressive effects on immune surveillance through functional impairment of the DC system.

Direct and indirect cross-tolerance of alloreactive T cells by dendritic cells retained in the immature stage.

BACKGROUND: Rejection of allografts entails the direct and indirect cross-recognition of donor major histocompatibility complex molecules by recipient alloreactive T cells. The ability to manipulate the state of dendritic cell (DC) maturation in vitro has enabled us to induce tolerance specifically targeting the alloreactive T-cell compartment. In this study, the immunoregulatory effect of alloantigen presentation by ex vivo-generated donor and recipient DCs retained in immature stage was investigated. METHODS: Dendritic cell were generated by culturing monocytes with granulocyte-macrophage colony-stimulating factor and interleukin-4. Ex vivo-generated tolerogenic DCs were characterized by flow cytometry and confocal microscopy. Recipient T-cell responses to donor or recipient DCs loaded with donor-derived apoptotic cells were assessed in a two-step culture system. RESULTS: Dendritic cells maintained their phagocytic and endocytic activities, and had significantly reduced capacity to prime recipient T cells. Moreover, primary coculture of recipient T cells with donor tolerogenic DCs rendered alloantigen-specific T cells hyporesponsive to a subsequent challenge with donor immunogenic DCs as evidenced by decreased proliferation and cytokine secretion. Importantly, recipient tolerogenic DCs loaded with donor-derived apoptotic cells were able to cross-tolerize recipient T cells. This was revealed by alloantigen-specific T-cell hyporesponsiveness on restimulation with the recipient immunogenic DCs loaded with different tissue-derived apoptotic cells obtained from the same donor. CONCLUSIONS: Dendritic cells retained in immature stage induce direct and most importantly indirect cross-tolerance of alloantigen-specific T cells. It may be likely that administration of donor and/or recipient DCs could be one means with which to promote tolerance induction in acute and chronic phases of organ transplant.