Indirect activation of naïve CD4+ T cells by dendritic cell-derived exosomes

Dendritic cells (DCs) secrete vesicles of endosomal origin, called exosomes, that bear major histocompatibility complex (MHC) and T cell costimulatory molecules. Here, we found that injection of antigen- or peptide-bearing exosomes induced antigen-specific na?ve CD4+ T cell activation in vivo. In vitro, exosomes did not induce antigen-dependent T cell stimulation unless mature CD8alpha- DCs were also present in the cultures. These mature DCs could be MHC class II-negative, but had to bear CD80 and CD86. Therefore, in addition to carrying antigen, exosomes promote the exchange of functional peptide-MHC complexes between DCs. Such a mechanism may increase the number of DCs bearing a particular peptide, thus amplifying the initiation of primary adaptive immune responses.     

A three-cell model for activation of naïve T helper cells

It is generally assumed that the activation of naïve T helper (Th) cells is the result of a two‐cell interaction between the Th cell and a dendritic cell (DC) and that three signals are required. Signal one or stimulation is the recognition by the T‐cell receptor (TCR) of antigenic peptides presented by major histocompatibility complex (MHC) class II molecules. Signal two or co‐stimulation is mainly provided by the triggering of CD28 on the T cell by CD80 and CD86 molecules on the DC. Signal three or polarization directs T‐cell differentiation into various effector phenotypes such as Th1 and Th2. Both signals, two and three, are often assumed to result from the binding of microbial products or endogenous molecular danger signals to germline‐encoded receptors such as toll‐like receptors (TLR) on the DC. However, recent data challenge this two‐cell model by revealing that Th1 polarization requires the presence of interferon‐γ (IFN‐γ) provided by a third cell. I propose here a three‐cell model for naïve Th‐cell activation. In this model, delivery of signal three by the DC is dependent on help provided by other innate immune cells such as NK cells, NK T cells, γδ T cells, mast cells, eosinophils and basophils. The rationale behind this model is that the innate immune system has been designed by evolution to select an appropriate class of immune response to protect the host.     

Gamma chain required for naïve CD4+ T cell survival but not for antigen proliferation

Lymphoid homeostasis is required to ensure immune responsiveness and to prevent immunodeficiency. As such, the immune system must maintain distinct populations of na?ve T cells that are able to respond to new antigens as well as memory T cells specific to those antigens it has already encountered. Though both na?ve and memory T cells reside in and traffic through secondary lymphoid organs, there is growing evidence that the two populations may be regulated differently. We show here that na?ve T cell survival and memory T cell survival have different requirements for cytokines (including the interleukins IL-2, IL-4, IL-7, IL-9 and IL-15) that use the common cytokine receptor gamma chain (gamma c). Using monoclonal populations of antigen-specific CD4+ T cells, we found that na?ve T cells cannot survive without gamma c, whereas memory T cells show no such requirement. In contrast, neither na?ve nor gamma c-deficient memory T cells were impaired in their ability to proliferate and produce cytokines in response to in vivo antigenic stimulation. These data call into question the physiological role of gamma c-dependent cytokines as T cell growth factors and show that na?ve and memory CD4+ T cell survival is maintained by distinct mechanisms.     

Differential regulation of naïve and memory CD4+ T cells by alternatively activated dendritic cells

Promising immunotherapeutic tools for T cell-mediated pathologies are alternatively activated dendritic cells (aaDC), which exert their effect through the regulation and tolerization of T cells. As na?ve and memory T cells have different susceptibilities to tolerogenic signals, it is important to understand the modulatory effects of aaDC on these T cell subsets. We have examined regulation of na?ve and memory CD4+ T cells by human aaDC generated with dexamethasone, the active form of vitamin D3, 1alpha,25-dihydroxyvitamin D3, and LPS. Although aaDC induced low, primary, allogeneic responses by na?ve and memory T cells, aaDC regulated the differentiation of these T cell subsets in a distinct manner. Na?ve T cells primed by aaDC retained a strong, proliferative capacity upon restimulation but were skewed toward a low IFN-gamma/high IL-10 cytokine profile. In contrast, memory T cells primed by aaDC became hyporesponsive in terms of proliferation and cytokine production. Induction of anergy in memory T cells by aaDC was not a result of the presence of CD25hi regulatory T cells and could be partially reversed by IL-2. Both T cell subsets acquired regulatory activity and inhibited primary CD4 and CD8 responses. Addition of exogenous IL-12p70 during T cell priming by aaDC prevented anergy induction in memory T cells and cytokine polarization in na?ve T cells, indicating that the lack of IL-12p70 is a key feature of aaDC. Our finding that aaDC differentially regulate na?ve and memory T cells is important for understanding and maximizing the therapeutic potential of aaDC.     

Vaccinia virus infection of mature dendritic cells results in activation of virus-specific naïve CD8+ T cells: a potential mechanism for direct presentation

Understanding how vaccinia virus (VV) generates immunity necessitates an appreciation for how this virus interacts with dendritic cells (DC), which are the most potent activators of na?ve CD8(+) T cells. In order to optimally activate na?ve CD8(+) T cells, DC must undergo maturation, during which costimulatory molecules are upregulated and cytokines are produced. In this report, we show that VV infection of immature murine bone marrow-derived DC (BMDC) failed to induce maturation. Similar results were obtained when CD8(+) DC were analyzed, a subset shown previously to be important in vivo in the generation of a vaccinia-specific response. The finding that VV infection of DC resulted in APC that were incapable of initiating T-cell activation was surprising given the previously reported role for direct presentation in the generation of anti-VV CD8(+) T-cell responses in mice. To address the potential mechanism responsible for direct presentation, we tested the hypothesis that previously matured DC were susceptible to vaccinia virus infection and could present newly synthesized VV-derived epitopes for CD8(+) T-cell activation. Our results show, that during VV infection of mature DC, threshold levels of viral protein are produced that promote T-cell activation. These results suggest that, even though VV cannot mature DC, previously matured DC exposed to VV can generate a VV-specific CD8(+) T-cell response providing a potential mechanism by which direct infection results in T-cell activation in vivo.     

Improved protocol for the isolation of naïve follicular dendritic cells

Follicular dendritic cells (FDCs) in lymphoid organs play an important role in the humoral immune response. However, because the isolation of FDCs is difficult due to their very small population size and fragility under mechanical and chemical stresses, the genetic and biochemical characteristics of FDCs remain unclear. Previously, we identified FDCs as ICAM-1⁺ cells in the CD45⁻ non-hematopoietic cell fraction from naïve mouse spleen after cell separation by means of digestion with a combination of enzymes. In the present study, using a new combination of enzymes, we found that FDCs are highly enriched in the CD45⁻ICAM-1⁺CD21/35⁺ cell fraction. CD45⁻ICAM-1⁺CD21/35⁺ cells in the mouse spleen retained an antigen administered in vivo for more than 7 days. Moreover, CD45⁻ICAM-1⁺CD21/35⁺ cells isolated from the spleen of mice administered with a cognate antigen enhanced the survival and proliferation of antigen-specific B cells in vitro. Our improved protocol for the isolation of naïve FDCs will be useful for the analysis of FDCs in vitro and in vivo.     

Response of naïve and memory CD8+ T cells to antigen stimulation in vivo

We studied the influence of memory T cell properties on the efficiency of secondary immune responses by comparing the in vivo immune response of the same numbers of T cell receptor (TCR) transgenic (Tg) na?ve and memory T cells. Compared to na?ve Tg cells, memory cells divided after a shorter lag time; had an increased division rate; a lower loss rate; and showed more rapid and efficient differentiation to effector functions. We found that initial na?ve T cell priming resulted in cells expressing mutually exclusive effector functions, whereas memory T cells were multifunctional after reactivation, with each individual cell expressing two to three different effector functions simultaneously. These special properties of memory T cells ensure the immediate control of reinfection.     

Interleukin-7 mediates the homeostasis of naïve and memory CD8 T cells in vivo

The na?ve and memory T lymphocyte pools are maintained through poorly understood homeostatic mechanisms that may include signaling via cytokine receptors. We show that interleukin-7 (IL-7) plays multiple roles in regulating homeostasis of CD8+ T cells. We found that IL-7 was required for homeostatic expansion of na?ve CD8+ and CD4+ T cells in lymphopenic hosts and for CD8+ T cell survival in normal hosts. In contrast, IL-7 was not necessary for growth of CD8+ T cells in response to a virus infection but was critical for generating T cell memory. Up-regulation of Bcl-2 in the absence of IL-7 signaling was impaired after activation in vivo. Homeostatic proliferation of memory cells was also partially dependent on IL-7. These results point to IL-7 as a pivotal cytokine in T cell homeostasis.     

Anti-ergotypic T cells in naïve rats

T regulatory cells play an important role in regulating T cell responses. Anti-ergotypic T cells are a subset of regulatory T cells that proliferate in response to activation markers, ergotopes, expressed on activated, and not on resting syngeneic T cells. Here we report the presence of anti-ergotypic T cells in lymph nodes, spleens and thymuses of naive rats. The development of anti-ergotypic T cells appeared to be independent of antigen priming, as thymocytes from one-day old rats exhibited significant anti-ergotypic proliferative responses. The anti-ergotypic T cells were found to be of the CD8+ phenotype, and included both TCRalpha/beta+ and TCRgamma/delta+ T cells. The TCRgamma/delta+ anti-ergotypic T cells secreted IFNgamma and TNFalpha in response to activated T cells; the TCRalpha/beta+ T cells proliferated but did not secret detectable cytokines. We found that the interaction between the anti-ergotypic T cells and stimulator T cells required cell-to-cell contact between the T cells. Professional APCs were not needed. The response of the TCRalpha/beta+CD8+ anti-ergotypic T cells was MHC-I restricted and B7-CD28 dependent; the response of the TCRgamma/delta+ anti-ergotypic T cells was B7-CD28 dependent, but was not inhibited by antibodies to classical MHC-I or MHC-II molecules. The existence of anti-ergotypic T cells in naive animals suggests that these cells might have a role in the regulation and maintenance of the immune system.     

IFN-α produced by human plasmacytoid dendritic cells enhances T cell-dependent naïve B cell differentiation

The development and quality of a humoral immune response are largely influenced by the environment that supports the activation of na?ve B cells. Human PDCs, through their unique capacity to produce high levels of IFN-α, have been shown earlier to enhance B cell responses stimulated by selected TLR ligands. In this study, we investigated whether PDCs also promote B cell activation induced by Th cell interactions and BCR ligation. Sorted human naive CD19(+) CD27(-) B cells were activated in vitro with anti-Ig and irradiated CD4(+) T cells. Under these conditions, the presence of supernatants from TLR-stimulated PDCs increased B cell proliferation, the frequency of B cells that differentiated to CD27(high) CD38(high) cells, and secretion of IgM. Similar results were observed when the B cells were activated in the presence of purified IFN-α. In contrast, supernatants from stimulated MDCs did not augment these functions. Also, IFN-α treatment of B cells up-regulated the expression of costimulatory molecule CD86 but not CD40, CD80, MHC class II, or CD25. Although direct IFN-α exposure of T cells suppressed their proliferative capacity, IFN-α treatment of B cells led to a small increase in their capacity to induce superantigen-driven activation of autologous CD4(+) T cells. In summary, PDCs, via their production of IFN-α, may render B cells more responsive to T cell contact, which in turn, facilitates B cell proliferation and differentiation to antibody-producing cells.     

Isolation and characterization of naïve follicular dendritic cells

Follicular dendritic cells (FDC) are specialized antigen-presenting cells to cognate B cells in the follicle of the lymphoid tissues. FDC also support survival and proliferation of the B cells, leading to the germinal center formation. FDC therefore play a central role in humoral immune responses. However, molecular and functional characteristics of FDC are largely unknown, because it is difficult to isolate and analyze FDC due to a very small number of FDC in the lymphoid tissues and the fragility by mechanical and chemical stresses in vitro. In this report, we established a novel method for FDC isolation from the spleen of na?ve mice by flow cytometry and analyzed the phenotypical and functional characteristics. The isolated FDC, which accounted for ~0.2% of the spleen cells of na?ve mice, were CD45(-), FDC-M2(+), and ICAM-1(+), and supported the survival and LPS-induced proliferation of B cells. We also showed that a neutralizing antibody against B cell activating factor TNF family (BAFF) suppressed FDC-dependent B cell proliferation in the presence of LPS, but not survival, demonstrating the evidence that FDC-derived BAFF is involved in B cell proliferation.     

Generation of anti-tumour effector T cells from naïve T cells by stimulation with dendritic/tumour fusion cells

Tumour-draining lymph node T cells are an excellent source of effector T cells that can be used in adoptive tumour immunotherapy because they have already been sensitized to tumour-associated antigens in vivo. However, such tumour-specific immune cells are not readily obtained from the host due to poor immunogenicity of tumours and reduced host immune responses. One obstacle in implementation of adoptive immunotherapy has been insufficient sensitization and expansion of tumour-specific effector cells. In this study, we aim to improve adoptive immunotherapy by generating anti-tumour effector T cells from naïve T lymphocytes. We attempted to achieve this by harnessing the advantages of dendritic cell (DC)-based anti-cancer vaccine strategies. Electrofusion was routinely employed to produce fusion cells with 30–40% efficiency by using the poorly immunogenic murine B16/F10 cell line, D5 cells, and DC generated from bone marrow cells. CD62L-positive T cells from spleens of naïve mice and the fusion cells were cocultured with a low concentration of IL-2. After 9 days of culture, the antigen-specific T cells were identified with an upregulation of CD25 and CD69 expression and a downregulation of CD62L expression. These cells secreted IFN-γ upon stimulation with irradiated tumour cells. Moreover, when transferred into mice with 3-day established pulmonary metastases, these cells with coadministration of IL-2 exhibited anti-tumour efficacy. In contrast, naïve T cells cocultured with a mixture of unfused DC and irradiated tumour cells did not exhibit anti-tumour efficacy. Our strategy provides the basis for a new approach in adoptive T cell immunotherapy for cancer.     

Transfer of macrophage-derived mycobacterial antigens to dendritic cells can induce naïve T-cell activation

Mycobacteria are capable of surviving and replicating in host macrophages, where they can release antigenic material into the environment. However, unlike dendritic cells (DCs), macrophages do not appear to be capable of activating naïve T cells. Therefore, this work investigated antigen transfer between macrophages and DCs. We generated culture supernatants from bacille Calmette–Guérin (BCG)-infected and uninfected macrophages and then determined whether DCs could present these extracellular mycobacterial antigens to T cells. Here, we show that DCs pulsed with antigens released from BCG-infected macrophages can stimulate primed T cells in vitro and initiate naïve T-cell responses in vivo. These results suggest that antigen transfer can occur between macrophages and DCs.     

Adoptive transfer of naïve dendritic cells in resolving post-sepsis long-term immunosuppression

Following initial episode of sepsis, survivors suffer an increased risk of mortality that persists long-term and remains a significant health care problem. Opportunistic infections and neoplasms are more frequent partially accounting for shorter life expectancy. This suggests that during sepsis the immune system becomes aberrant and is unable to restore its optimal function after the initial insult. Dendritic cells (DC) or their precursors are frequently affected during the initial episode of sepsis as well as after the resolution of symptom. Considering these cells’ pivotal role in regulating innate and acquired immune, they are promising candidates for therapeutic manipulation. Sepsis induces several changes in the populations of DCs via epigenetic, and/or other, mechanisms. Here, we propose that transplantation of ‘naïve dendritic cells’ (DCs which are never exposed to sepsis) can reverse several aspects of the long-term post-sepsis immunosuppression. Furthermore, we present advantages of the presented therapeutic approach.     

Negative regulators in homeostasis of naïve peripheral T cells

It is now apparent that naïve peripheral T cells are a dynamic population where active processes prevent inappropriate activation while supporting survival. The process of thymic education makes naïve peripheral T cells dependent on interactions with self-MHC for survival. However, as these signals can potentially result in inappropriate activation, various non-redundant, intrinsic negative regulatory molecules including Tob, Nfatc2, and Smad3 actively enforce T cell quiescence. Interactions among these pathways are only now coming to light and may include positive or negative crosstalk. In the case of positive crosstalk, self-MHC initiated signals and intrinsic negative regulatory factors may cooperate to dampen T cell activation and sustain peripheral tolerance in a binary fashion (on–off). In the case of negative crosstalk, self-MHC signals may promote survival through partial activation while intrinsic negative regulatory factors act as rheostats to restrain cell cycle entry and prevent T cells from crossing a threshold that would break tolerance.