Laura C. Bonifaz

Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity

Induction of immunity and peripheral tolerance requires contacts between antigen-bearing dendritic cells (DCs) and cognate T cells. Using real-time two-photon microscopy, we have analyzed the dynamics of CD8+ T cells in lymph nodes during the induction of antigen-specific immunity or tolerance. At 15–20 h after the induction of immunity, T cells stopped moving and established prolonged interactions with DCs. In tolerogenic conditions, despite effective initial T cell activation and proliferation, naive T cells remained motile and established serial brief contacts with multiple DCs. Thus, stable DC–T cell interactions occur during the induction of priming, whereas brief contacts may contribute to the induction of T cell tolerance.

Endogenous and exogenous forms of the same antigen are processed from different pools to bind MHC class II molecules in endocytic compartments

The current studies were carried out to examine the basis for the differences in the antigenic peptides generated from exogenous and endogenous forms of hen egg white lysozyme (HEL). The role of different intracellular compartments in the generation and binding of HEL peptides derived from two endogenous forms of HEL, either secreted (sHEL) or retained in the endoplasmic reticulum (ER, KDELHEL), presented by MHC class II molecules was examined and compared to exogenous HEL. Initially it was found that antigen‐presenting cells bearing both intracellular forms of HEL generated and presented a number of IAk‐restricted HEL epitopes to T cell hybridomas, although s HEL was processed more efficiently than KDELHEL. There were differences, however, for some determinants between endogeneous and exogenous HEL. At equivalent antigen‐presenting efficiencies, endogenous HEL‐bearing cells displayed a lower surface density of IAk‐bound HEL‐52‐61‐related peptides than cells pulsed with exogenous HEL, as detected by a specific monoclonal antibody. Neither endogenous HEL degradation nor peptide binding to MHC class II molecules occurred in the ER. Processing of sHEL and KDELHEL appears to take place either in a post‐trans‐Golgi network acidic compartment or in the cytosol, whereas peptide binding to MHC class II molecules occurs in endocytic compartments . Furthermore, the peptides generated were derived from an endogenous source rather than from secreted and re‐endocytosed HEL. Thus, processing of endogenous HEL is from a different pool than exogenous HEL and occurs in different compartments.

Tumor cells prevent mouse dendritic cell maturation induced by TLR ligands.

Tumor cells can evade the immune system through several mechanisms, one of which is to block DC maturation. It has been suggested that signaling via Toll-like receptors (TLR) may be involved in the induction of prophylactic anti-cancer immunity and in the treatment of established tumors. In the present study we found that high numbers of tumor cells interfere with BMDC activation induced by the TLR ligands LPS and poly IC. Tumor cells blocked TLR3- and TLR4-mediated induction of MHCII and the co-stimulatory molecules CD40 and CD86, as well as the cytokines IL-12, TNF-α and IL-6. Importantly, tumor cells induced inhibitory molecules (B7-DC, B7-H1 and CD80) on spleen DC in vivo and on BMDC, even in the presence of TLR ligands. Moreover, after a long exposure with tumor cells, purified BMDC were unable to respond to a second challenge with TLR ligands. The failure of tumor exposed-BMDC to express co-stimulatory molecules and cytokines in the presence of TLR ligands has implications for the future development of DC-based cancer immune therapies using TLR ligands as adjuvants for the activation of DC.

CD40-Induced Aggregation of MHC Class II and CD80 on the Cell Surface Leads to an Early Enhancement in Antigen Presentation

Ligation of CD40 on B cells increases their ability to present Ag and to activate MHC class II (MHC-II)-restricted T cells. How this occurs is not entirely clear. In this study we demonstrate that CD40 ligation on Ag-presenting B cells (APC) for a short period between 30 min and 3 h has a rapid, augmenting effect on the ability of a B cell line and normal B cells to activate T cells. This is not due to alterations in Ag processing or to an increase in surface expression of CD80, CD86, ICAM-1, or MHC-II. This effect is particularly evident with naive, resting T lymphocytes and appears to be more pronounced under limiting Ag concentrations. Shortly after CD40 ligation on a B cell line, MHC-II and CD80 progressively accumulated in cholesterol-enriched microdomains on the cell surface, which correlated with an initial enhancement in their Ag presentation ability. Moreover, CD40 ligation induced a second, late, more sustained enhancement of Ag presentation, which correlates with a significant increase in CD80 expression by APC. Thus, CD40 signaling enhances the efficiency with which APC activate T cells by at least two related, but distinct, mechanisms: an early stage characterized by aggregation of MHC-II and CD80 clusters, and a late stage in which a significant increase in CD80 expression is observed. These results raise the possibility that one important role of CD40 is to contribute to the formation of the immunological synapse on the APC side.

Helminth-excreted/secreted products are recognized by multiple receptors on DCs to block the TLR response and bias Th2 polarization in a cRAF dependent pathway

Dendritic cells (DCs) recognize pathogens and initiate the T‐cell response. The DC‐helminth interaction induces an immature phenotype in DCs; as a result, these DCs display impaired responses to TLR stimulation and prime Th2‐type responses. However, the DC receptors and intracellular pathways targeted by helminth molecules and their importance in the initiation of the Th2 response are poorly understood. In this report, we found that products excreted/secreted by Taenia crassiceps (TcES) triggered cRAF phosphorylation through MGL, MR, and TLR2. TcES interfered with the LPS‐induced NFκB p65 and p38 MAPK signaling pathways. In addition, TcES‐induced cRAF signaling pathway was critical for down‐regulation of the TLR‐mediated DC maturation and secretion of IL‐12 and TNF‐α. Finally, we show for the first time that blocking cRAF in DCs abolishes their ability to induce Th2 polarization in vitro after TcES exposure. Our data demonstrate a new mechanism by which helminths target intracellular pathways to block DC maturation and efficiently program Th2 polarization.—Terrazas, C. A., Alcántara‐Hernández, M., Bonifaz, L., Terrazas, L. I., Satoskar, A. R. Helminth‐excreted/secreted products are recognized by multiple receptors on DCs to block the TLR response and bias Th2 polarization in a cRAF dependent pathway. FASEB J. 27, 4547–4560 (2013). www.fasebj.org

Heat shock protein 70 down-regulates the production of toll-like receptor-induced pro-inflammatory cytokines by a heat shock factor-1/constitutive heat shock element-binding factor-dependent mechanism

BackgroundHeat shock protein 70 (Hsp70) is an intracellular chaperone protein with regulatory and cytoprotective functions. Hsp70 can also be found in the extracellular milieu, as a result of active secretion or passive release from damaged cells. The role of extracellular Hsp70 is not fully understood. Some studies report that it activates monocytes, macrophages and dendritic cells through innate immune receptors (such as Toll-like receptors, TLRs), while others report that Hsp70 is a negative regulator of the inflammatory response. In order to address this apparent inconsistency, in this study we evaluated the response of human monocytes to a highly purified recombinant Hsp70.MethodsHuman peripheral blood monocytes were stimulated with Hsp70, alone or in combination with TLR agonists. Cytokines were quantified in culture supernatants, their mRNAs were measured by RT-PCR, and the binding of transcription factors was evaluated by electrophoretic mobility shift assay (EMSA). Kruskal-Wallis test or one-way or two-way ANOVA were used to analyze the data.ResultsThe addition of Hsp70 to TLR-activated monocytes down-regulated TNF-α as well as IL-6 levels. This effect was independent of a physical interaction between Hsp70 and TLR agonists; instead it resulted of changes at the TNF-α gene expression level. The decrease in TNF-α expression correlated with the binding of HSF-1 (heat shock transcription factor 1, a transcription factor activated in response to Hsp70) and CHBF (constitutive HSE-binding factor) to the TNF-α gene promoter.ConclusionExtracellular Hsp70 negatively regulates the production of pro-inflammatory cytokines of monocytes exposed to TLR agonists and contributes to dampen the inflammatory response.

Intradermal immunization in the ear with cholera toxin and its non-toxic β subunit promotes efficient Th1 and Th17 differentiation dependent on migrating DCs.

The nature of CD4+ T‐cell responses after skin immunization and the role of migrating DCs in the presence of adjuvants in the elicited response are interesting issues to be investigated. Here, we evaluated the priming of CD4+ T cells following ear immunization with low doses of model antigens in combination with either cholera toxin (CT) or the non‐toxic β CT subunit (CTB) as an adjuvant. Following immunization with CT, we found efficient antigen presentation that is reflected in the production of IFN‐γ and IL‐17 by CD4+ T cells over IL‐4 or IL‐5 production. The CTB‐induced activation of DCs in the ear occurred without visible inflammation, which reflects a similar type of CD4+ T‐cell differentiation. In both cases, the elicited response was dependent on the presence of migrating skin cells. Remarkably, immunization with CT or with CTB led to the induction of a delayed‐type hypersensitivity (DTH) response in the ear. The DTH response that was induced by CT immunization was dependent on IL‐17 and partially dependent on IFN‐γ activity. These results indicate that both CT and CTB induce an efficient CD4+ T‐cell response to a co‐administered antigen following ear immunization that is dependent on migrating DCs.

Salmonella Typhi OmpS1 and OmpS2 porins are potent protective immunogens with adjuvant properties

Salmonella enterica serovar Typhi (S. Typhi) is the causal agent of typhoid fever, a disease that primarily affects developing countries. Various antigens from this bacterium have been reported to be targets of the immune response. Recently, the S. Typhi genome has been shown to encode two porins – OmpS1 and OmpS2 – which are expressed at low levels under in vitro culture conditions. In this study, we demonstrate that immunizing mice with either OmpS1 or OmpS2 induced production of specific, long‐term antibody titres and conferred protection against S. Typhi challenge; in particular, OmpS1 was more immunogenic and conferred greater protective effects than OmpS2. We also found that OmpS1 is a Toll‐like receptor 4 (TLR4) agonist, whereas OmpS2 is a TLR2 and TLR4 agonist. Both porins induced the production of tumour necrosis factor and interleukin‐6, and OmpS2 was also able to induce interleukin‐10 production. Furthermore, OmpS1 induced the over‐expression of MHC II molecules in dendritic cells and OmpS2 induced the over‐expression of CD40 molecules in macrophages and dendritic cells. Co‐immunization of OmpS1 or OmpS2 with ovalbumin (OVA) increased anti‐OVA antibody titres, the duration and isotype diversity of the OVA‐specific antibody response, and the proliferation of T lymphocytes. These porins also had adjuvant effects on the antibody response when co‐immunized with either the Vi capsular antigen from S. Typhi or inactivated 2009 pandemic influenza A(H1N1) virus [A(H1N1)pdm09]. Taken together, the data indicate that OmpS1 and OmpS2, despite being expressed at low levels under in vitro culture conditions, are potent protective immunogens with intrinsic adjuvant properties.

PD-L2 induction on dendritic cells exposed to Mycobacterium avium downregulates BCG-specific T cell response.

The exposure to certain species of Nontuberculous Mycobacteria (NTM) can modulate the immune response induced by Mycobacterium bovis BCG. Mycobacterium avium has been postulated as a weak inducer of dendritic cell (DC) maturation. However, how the DC exposure to M. avium could contribute to the modulation of a BCG-specific CD4+ T cell response and the molecules involved remain unknown. Here, we exposed bone marrow-derived DCs (BMDCs) to M. avium either prior to exposure to BCG or as a unique stimulus. We found that M. avium induces high expression of PD-L2 (B7-DC) in BMDCs. This was dependent on IL-10 production through the TLR2-p38 MAPK signaling pathway. Exposure to M. avium prior to BCG results in BMDCs that do not express co-stimulatory molecules and pro-inflammatory cytokines, while the expression of PD-L2 and IL-10 was maintained. BMDCs exposed to M. avium impaired the activation of BCG-specific T cells through the PD-1: PD-L interaction. This suggests that a M. avium-induced phenotype in DCs might be implicated in the induction of mechanisms of tolerance that could impact the T cell response induced by BCG vaccination.

Antibody Persistence in Adults Two Years after Vaccination with an H1N1 2009 Pandemic Influenza Virus-Like Particle Vaccine.

The influenza virus is a human pathogen that causes epidemics every year, as well as potential pandemic outbreaks, as occurred in 2009. Vaccination has proven to be sufficient in the prevention and containment of viral spreading. In addition to the current egg-based vaccines, new and promising vaccine platforms, such as cell culture-derived vaccines that include virus-like particles (VLPs), have been developed. VLPs have been shown to be both safe and immunogenic against influenza infections. Although antibody persistence has been studied in traditional egg-based influenza vaccines, studies on antibody response durations induced by VLP influenza vaccines in humans are scarce. Here, we show that subjects vaccinated with an insect cell-derived VLP vaccine, in the midst of the 2009 H1N1 influenza pandemic outbreak in Mexico City, showed antibody persistence up to 24 months post-vaccination. Additionally, we found that subjects that reported being revaccinated with a subsequent inactivated influenza virus vaccine showed higher antibody titres to the pandemic influenza virus than those who were not revaccinated. These findings provide insights into the duration of the antibody responses elicited by an insect cell-derived pandemic influenza VLP vaccine and the possible effects of subsequent influenza vaccination on antibody persistence induced by this VLP vaccine in humans.