The genetic background influences the cellular and humoral immune responses to vaccines.

The assessment of Toll-like receptor (TLR) agonists as candidate adjuvants for induction of effective T helper type 1 (Th1) immunity continues to rely on the use of mice. However, the genetic variation among inbred mice may influence the efficacy of adjuvants and bias a study's conclusions. Here, we evaluated the differences in cellular and humoral responses of genetically non-identical mouse strains immunized with ovalbumin (OVA) plus alum, TLR-3, TLR-4, TLR-7/8 or TLR-9 agonists. We found that all the tested TLR agonists recruited dendritic cells (DCs) and natural killer (NK) cells significantly into the lymph nodes, promoted DC-NK cross-talk and enhanced the cellular responses in B6 strain. In contrast, TLR-3 and TLR-7/8 were the only two agonists that showed the cellular adjuvanticity in the BALB/c strain. Compared with other TLR agonists, TLR-3 and TLR-7/8 were demonstrated to be the most effective adjuvants to generate interferon (IFN)-γ-producing effector NK, CD4, and CD8 T cells in B6 and BALB/c strains, respectively. We also found that compared with alum, all adjuvants induced the recruitment of B cells and production of OVA-specific immunoglobulin (Ig)G2a more effectively in both strains. In addition, the B6 strain recruited more B cells, but surprisingly produced significantly lower amounts of OVA-specific IgG2a in response to all adjuvants. However, consistent with the frequency of IFN-γ-producing effector cells observed in individual strains following immunizations, we detected more OVA-specific IgG2a in serum of B6 and BALB/c strains in response to TLR-3 and TLR-7/8, respectively. Our data suggest that genetic background should be taken into consideration when evaluating the activities of TLR agonists for the development of prophylactic and therapeutic vaccines.

Nicotine exposure alters the mRNA expression of Notch ligands in dendritic cells and their response to Th1-/Th2-promoting stimuli.

Dendritic cells (DCs) utilize polarizing signals to instruct the differentiation of T helper (Th) cells into Th1 and Th2 effector cells: antigen-specific 'signal 1', costimulatory 'signal 2' and polarizing cytokines 'signal 3'. Accumulating evidence suggests the involvement of an additional signal, the Notch signalling pathway. We reported that in response to Th1-promoting stimuli, both mouse and human DCs generated in the presence of the immune modulator nicotine (nicDCs) fail to support the development of effector memory Th1 cells. However, in response to Th2-promoting stimuli, these nicDCs preferentially support the differentiation of antigen-specific IL-4-producing Th2 effector cells. Here, we show that when compared to their control counterparts, immature mouse and human nicDCs display higher levels of the Notch ligands D1, D4 and J2 mRNA expression. In response to Th1- and Th2-promoting stimuli, mouse nicDCs display higher levels of the Notch ligands D1, D4 and J2, while human nicDCs show higher levels of D1, D4 and J1 mRNA expression. Furthermore, both stimulated mouse and human nicDCs express higher CD86 to CD80 ratio and produce lower amount of IL-12. Collectively, our data suggest that these changes in addition to an increase in Jagged expression correlate with the ability of nicDCs to modulate the Th1/Th2 balance in favour of Th2 generation.

The BCL-6 proto-oncogene controls germinal-centre formation and Th2-type inflammation.

Structural alterations of the promoter region of the BCL-6 proto-oncogene represent the most frequent genetic alteration associated with non-Hodgkin lymphoma, a malignancy often deriving from germinal-centre B cells. The BCL-6 gene encodes a zinc-finger transcriptional repressor normally expressed in both B cells and CD4+ T cells within germinal centres, but its precise function is unknown. We show that mice deficient in BCL-6 displayed normal B-cell, T-cell and lymphoid-organ development but have a selective defect in T-cell-dependent antibody responses. This defect included a complete lack of affinity maturation and was due to the inability of follicular B cells to proliferate and form germinal centres. In addition, BCL-6-deficient mice developed an inflammatory response in multiple organs characterized by infiltrations of eosinophils and IgE-bearing B lymphocytes typical of a Th2-mediated hyperimmune response. Thus, BCL-6 functions as a transcriptional switch that controls germinal centre formation and may also modulate specific T-cell-mediated responses. Altered expression of BCL-6 in lymphoma represents a deregulation of the pathway normally leading to B cell proliferation and germinal centre formation.

Cross-priming of naive CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells.

The goal of tumor immunotherapy is to elicit immune responses against autologous tumors. It would be highly desirable that such responses include multiple T cell clones against multiple tumor antigens. This could be obtained using the antigen presenting capacity of dendritic cells (DCs) and cross-priming. That is, one could load the DC with tumor lines of any human histocompatibility leukocyte antigen (HLA) type to elicit T cell responses against the autologous tumor. In this study, we show that human DCs derived from monocytes and loaded with killed melanoma cells prime naive CD45RA(+)CD27(+)CD8(+) T cells against the four shared melanoma antigens: MAGE-3, gp100, tyrosinase, and MART-1. HLA-A201(+) naive T cells primed by DCs loaded with HLA-A201(-) melanoma cells are able to kill several HLA-A201(+) melanoma targets. Cytotoxic T lymphocyte priming towards melanoma antigens is also obtained with cells from metastatic melanoma patients. This demonstration of cross-priming against shared tumor antigens builds the basis for using allogeneic tumor cell lines to deliver tumor antigens to DCs for vaccination protocols.

Nicotine alters the biological activities of developing mouse bone marrow-derived dendritic cells (DCs).

Cigarette smoke contains nicotine, an immunomodulatory component that is thought to affect immune surveillance and increase the progression of diseases. Dendritic cells (DCs) constitute a family of antigen-presenting (APCs) with inherent abilities to sense and translate environmental cues and to shape host immunity. We recently reported on the effects of nicotine on human DCs and proposed a possible mechanism that links cigarette smoke to higher incidences of respiratory tract infection and asthma. To establish the causal relationship between nicotine-induced DC alterations and immunomodulation in vivo, we translated our in vitro human results to the mouse system and studied the direct effects of nicotine exposure on the biological and functional properties of mouse bone marrow (BM) DCs differentiated in vitro from their precursors. We report that while the presence of nicotine in the microenvironment has no direct effect on competent mouse BM-derived DCs function, it promotes the development of mouse BM DC precursors into DCs (thereafter called nicDCs) with a semi-mature phenotype revealed by higher expression of costimulatory molecules CD80, CD86, CD40, MHC II molecules and the lymph node homing receptor, CCR7. Consistent with their maturational status, these nicDCs have reduced capacity for antigen uptake and produce substantially less Th1-promoting cytokine, IL-12, in response to Th1-polarizing adjuvant, lipopolysaccharide (LPS). Interestingly, we found that nicDCs preferentially support the proliferation and differentiation of ovalbumin (OVA)-specific naïve T cells into effector memory cells, producing significantly less IFN-gamma and more IL-4. These results provide evidence for the similarity in the effects of nicotine on mouse and human DCs, particularly the ability to modulate DC differentiation towards developing Th2 immunity.

Dendritic cell based tumor vaccines.

Dendritic cells (DC) constitute a unique system of cells that induce, sustain and regulate immune responses. Distributed as sentinels throughout the body, DC are poised to capture antigen (Ag), migrate to draining lymphoid organs, and, after a process of maturation, select Ag-specific lymphocytes to which they present the processed Ag, thereby inducing immune responses. DC present Ag to CD4(+) T cells which in turn regulate multiple effectors, including CD8(+) cytotoxic T cells, B cells, NK cells, macrophages and eosinophils, all of which contribute to the protective immune responses. Several key features of the DC system may be highlighted: (1) the existence of different DC subsets that share biological functions, yet display unique ones such as polarization of T cell responses towards Type 1 or Type 2 or regulation of B cell responses; (2) the functional specialization of DC according to their differentiation/maturation stages; and (3) the plasticity of DC which is determined by the microenvironment (e.g. cytokines) and may manifest as (i) the final differentiation into either DC (enhanced antigen presentation) or macrophage (enhanced antigen degradation); (ii) the induction of immunity or tolerance; and (iii) the polarization of T cell responses. Because of these unique properties, DC represent both vectors and targets for immunological intervention in numerous diseases and are optimal candidates for vaccination protocols both in cancer and infectious diseases.

Purification of rabbit blood basophils by isopycnic banding on Percoll.

This study defines the basophil densities in rabbits and also describes a relatively simple method of purifying basophils from peripheral blood through a two-step procedure. To evaluate the recovery of basophils, EDTA-anticoagulated blood in layered over Percoll of various densities, ranging from 1.070 to 1.080 g/ml. The cells retained in Percoll were collected separately after centrifugation at 600 g(av) for 30 min (Step I). Distribution of basophils indicated that basophils had densities of between 1.070 and 1.078 g/ml, with a peak at 1.074 g/ml. To enhance the basophil purity, a second centrifugation of the cells obtained from Step I over appropriate Percoll gradient led to further enhancement of basophil purity (30.4 +/- 0.7%) which was (20.6 +/- 0.2%) in Step I. The cells obtained by this method appeared morphologically normal and viable.

Dendritic cells capture killed tumor cells and present their antigens to elicit tumor-specific immune responses.

Due to their capacity to induce primary immune responses, dendritic cells (DC) are attractive vectors for immunotherapy of cancer. Yet the targeting of tumor Ags to DC remains a challenge. Here we show that immature human monocyte-derived DC capture various killed tumor cells, including Jurkat T cell lymphoma, malignant melanoma, and prostate carcinoma. DC loaded with killed tumor cells induce MHC class I- and class II-restricted proliferation of autologous CD8+ and CD4+ T cells, demonstrating cross-presentation of tumor cell-derived Ags. Furthermore, tumor-loaded DC elicit expansion of CTL with cytotoxic activity against the tumor cells used for immunization. CTL elicited by DC loaded with the PC3 prostate carcinoma cell bodies kill another prostate carcinoma cell line, DU145, suggesting recognition of shared Ags. Finally, CTL elicited by DC loaded with killed LNCap prostate carcinoma cells, which express prostate specific Ag (PSA), are able to kill PSA peptide-pulsed T2 cells. This demonstrates that induced CTL activity is not only due to alloantigens, and that alloantigens do not prevent the activation of T cells specific for tumor-associated Ags. This approach opens the possibility of using allogeneic tumor cells as a source of tumor Ag for antitumor therapies.

Mature dendritic cells infiltrate the T cell-rich region of oral mucosa in chronic periodontitis: in situ, in vivo, and in vitro studies.

Previous studies have analyzed the lymphoid and myeloid foci within the gingival mucosa in health and chronic periodontitis (CP); however, the principal APCs responsible for the formation and organizational structure of these foci in CP have not been defined. We show that in human CP tissues, CD1a(+) immature Langerhans cells predominantly infiltrate the gingival epithelium, whereas CD83(+) mature dendritic cells (DCs) specifically infiltrate the CD4(+) lymphoid-rich lamina propria. In vivo evidence shows that exacerbation of CP results in increased levels of proinflammatory cytokines that mediate DC activation/maturation, but also of counterregulatory cytokines that may prevent a Th-polarized response. Consistently, in vitro-generated monocyte-derived DCs pulsed with Porphyromonas gingivalis strain 381 or its LPS undergo maturation, up-regulate accessory molecules, and release proinflammatory (IL-1beta, PGE(2)) and Th (IL-10, IL-12) cytokines. Interestingly, the IL-10:IL-12 ratio elicited from P. gingivalis-pulsed DCs was 3-fold higher than that from Escherichia coli-pulsed DCs. This may account for the significantly (p < 0.05) lower proliferation of autologous CD4(+) T cells and reduced release of IFN-gamma elicited by P. gingivalis-pulsed DCs. Taken together, these findings suggest a previously unreported mechanism for the pathophysiology of CP, involving the activation and in situ maturation of DCs by the oral pathogen P. gingivalis, leading to release of counterregulatory cytokines and the formation of T cell-DC foci.