Functional diversity of human vaginal APC subsets in directing T-cell responses.

Human vaginal mucosa is the major entry site of sexually transmitted pathogens and thus has long been attractive as a site for mounting mucosal immunity. It is also known as a tolerogenic microenvironment. Here, we demonstrate that immune responses in the vagina can be orchestrated by the functional diversity of four major antigen-presenting cell (APC) subsets. Langerhans cells (LCs) and CD14(-) lamina propria-dendritic cells (LP-DCs) polarize CD4(+) and CD8(+) T cells toward T-helper type 2 (Th2), whereas CD14(+) LP-DCs and macrophages polarize CD4(+) T cells toward Th1. Both LCs and CD14(-) LP-DCs are potent inducers of Th22. Owing to their functional specialties and the different expression levels of pattern-recognition receptors on the APC subsets, microbial products do not bias them to elicit common types of immune responses (Th1 or Th2). To evoke desired types of adaptive immune responses in the human vagina, antigens may need to be targeted to proper APC subsets with right adjuvants.

Dendritic cells and immunity against cancer.

T cells can reject established tumours when adoptively transferred into patients, thereby demonstrating the power of the immune system for cancer therapy. However, it has proven difficult to maintain adoptively transferred T cells in the long term. Vaccines have the potential to induce tumour-specific effector and memory T cells. However, clinical efficacy of current vaccines is limited, possibly because tumours skew the immune system by means of myeloid-derived suppressor cells, inflammatory type 2 T cells and regulatory T cells (Tregs), all of which prevent the generation of effector cells. To improve the clinical efficacy of cancer vaccines in patients with metastatic disease, we need to design novel and improved strategies that can boost adaptive immunity to cancer, help overcome Tregs and allow the breakdown of the immunosuppressive tumour microenvironment. This can be achieved by exploiting the fast increasing knowledge about the dendritic cell (DC) system, including the existence of distinct DC subsets that respond differentially to distinct activation signals, (functional plasticity), both contributing to the generation of unique adaptive immune responses. We foresee that these novel cancer vaccines will be used as monotherapy in patients with resected disease and in combination with drugs targeting regulatory/suppressor pathways in patients with metastatic disease.

Assessing oncologic benefit in clinical trials of immunotherapy agents.

BACKGROUND: USA Food and Drug Administration approval for cancer therapy requires demonstration of patient benefit as a marker of clinical efficacy. Prolonged survival is the gold standard for demonstration of efficacy, but other end points such as antitumor response, progression-free survival, quality of life, or surrogate end points may be used. DESIGN: This study was developed based on discussion during a roundtable meeting of experts in the field of immunotherapy. RESULTS: In most clinical trials involving cytotoxic agents, response end points use RECIST based on the premise that 'effective' therapy causes tumor destruction, target lesion shrinkage, and prevention of new lesions. However, RECIST may not be appropriate in trials of immunotherapy. Like other targeted agents, immunotherapies may mediate cytostatic rather than direct cytotoxic effects, and these may be difficult to quantify with RECIST. Furthermore, significant time may elapse before clinical effects are quantifiable because of complex response pathways. Effective immunotherapy may even mediate transient lesion growth secondary to immune cell infiltration. CONCLUSIONS: RECIST may not be an optimal indicator of clinical benefit in immunotherapy trials. This article discusses alternative clinical trial designs and end points that may be more relevant for immunotherapy trials and may offer more effective prediction of survival in pivotal phase III studies.

Induction of dendritic cell differentiation by IFN-alpha in systemic lupus erythematosus.

Dendritic cells (DCs) are important in regulating both immunity and tolerance. Hence, we hypothesized that systemic lupus erythematosus (SLE), an autoimmune disease characterized by autoreactive B and T cells, may be caused by alterations in the functions of DCs. Consistent with this, monocytes from SLE patients' blood were found to function as antigen-presenting cells, in vitro. Furthermore, serum from SLE patients induced normal monocytes to differentiate into DCs. These DCs could capture antigens from dying cells and present them to CD4-positive T cells. The capacity of SLE patients' serum to induce DC differentiation correlated with disease activity and depended on the actions of interferon-alpha (IFN-alpha). Thus, unabated induction of DCs by IFN-alpha may drive the autoimmune response in SLE.

Interleukin 15 skews monocyte differentiation into dendritic cells with features of Langerhans cells.

Langerhans cells (LCs) represent a subset of immature dendritic cells (DCs) specifically localized in the epidermis and other mucosal epithelia. As surrounding keratinocytes can produce interleukin (IL)-15, a cytokine that utilizes IL-2Rgamma chain, we analyzed whether IL-15 could skew monocyte differentiation into LCs. Monocytes cultured for 6 d with granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-15 differentiate into CD1a(+)HLA-DR(+)CD14(-)DCs (IL15-DCs). Agents such as lipopolysaccharide (LPS), tumor necrosis factor (TNF)alpha, and CD40L induce maturation of IL15-DCs to CD83(+), DC-LAMP(+) cells. IL15-DCs are potent antigen-presenting cells able to induce the primary (mixed lymphocyte reaction [MLR]) and secondary (recall responses to flu-matrix peptide) immune responses. As opposed to cultures made with GM-CSF/IL-4 (IL4-DCs), a proportion of IL15-DCs expresses LC markers: E-Cadherin, Langerin, and CC chemokine receptor (CCR)6. Accordingly, IL15-DCs, but not IL4-DCs, migrate in response to macrophage inflammatory protein (MIP)-3alpha/CCL20. However, IL15-DCs cannot be qualified as "genuine" Langerhans cells because, despite the presence of the 43-kD Langerin, they do not express bona fide Birbeck granules. Thus, our results demonstrate a novel pathway in monocyte differentiation into dendritic cells.

Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo.

The adaptive immune system has evolved distinct responses against different pathogens, but the mechanism(s) by which a particular response is initiated is poorly understood. In this study, we investigated the type of Ag-specific CD4(+) Th and CD8(+) T cell responses elicited in vivo, in response to soluble OVA, coinjected with LPS from two different pathogens. We used Escherichia coli LPS, which signals through Toll-like receptor 4 (TLR4) and LPS from the oral pathogen Porphyromonas gingivalis, which does not appear to require TLR4 for signaling. Coinjections of E. coli LPS + OVA or P. gingivalis LPS + OVA induced similar clonal expansions of OVA-specific CD4(+) and CD8(+) T cells, but strikingly different cytokine profiles. E. coli LPS induced a Th1-like response with abundant IFN-gamma, but little or no IL-4, IL-13, and IL-5. In contrast, P. gingivalis LPS induced Th and T cell responses characterized by significant levels of IL-13, IL-5, and IL-10, but lower levels of IFN-gamma. Consistent with these results, E. coli LPS induced IL-12(p70) in the CD8alpha(+) dendritic cell (DC) subset, while P. gingivalis LPS did not. Both LPS, however, activated the two DC subsets to up-regulate costimulatory molecules and produce IL-6 and TNF-alpha. Interestingly, these LPS appeared to have differences in their ability to signal through TLR4; proliferation of splenocytes and cytokine secretion by splenocytes or DCs from TLR4-deficient C3H/HeJ mice were greatly impaired in response to E. coli LPS, but not P. gingivalis LPS. Therefore, LPS from different bacteria activate DC subsets to produce different cytokines, and induce distinct types of adaptive immunity in vivo.

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.

Increased frequency of pre-germinal center B cells and plasma cell precursors in the blood of children with systemic lupus erythematosus.

We have analyzed the blood B cell subpopulations of children with systemic lupus erythematosus (SLE) and healthy controls. We found that the normal recirculating mature B cell pool is composed of four subsets: conventional naive and memory B cells, a novel B cell subset with pregerminal center phenotype (IgD(+)CD38(+)centerin(+)), and a plasma cell precursor subset (CD20(-)CD19(+/low)CD27(+/++) CD38(++)). In SLE patients, naive and memory B cells (CD20(+)CD38(-)) are approximately 90% reduced, whereas oligoclonal plasma cell precursors are 3-fold expanded, independently of disease activity and modality of therapy. Pregerminal center cells in SLE are decreased to a lesser extent than conventional B cells, and therefore represent the predominant blood B cell subset in a number of patients. Thus, SLE is associated with major blood B cell subset alterations.

Immune and clinical responses in patients with metastatic melanoma to CD34(+) progenitor-derived dendritic cell vaccine.

Immunization to multiple defined tumor antigens for specific immune therapy of human cancer has thus far proven difficult. Eighteen HLA A*0201(+) patients with metastatic melanoma received injections s.c. of CD34(+)progenitor-derived autologous dendritic cells (DCs), which included Langerhans cells. DCs were pulsed with peptides derived from four melanoma antigens [(MelAgs) MelanA/MART-1, tyrosinase, MAGE-3, and gp100], as well as influenza matrix peptide (Flu-MP) and keyhole limpet hemocyanin (KLH) as control antigens. Overall immunological effects were assessed by comparing response profiles using marginal likelihood scores. DC injections were well tolerated except for progressive vitiligo in two patients. DCs induced an immune response to control antigens (KLH, Flu-MP) in 16 of 18 patients. An enhanced immune response to one or more MelAgs was seen in these same 16 patients, including 10 patients who responded to >2 MelAgs. The two patients failing to respond to both control and tumor antigens experienced rapid tumor progression. Of 17 patients with evaluable disease, 6 of 7 patients with immunity to two or less MelAgs had progressive disease 10 weeks after study entry, in contrast to tumor progression in only 1 of 10 patients with immunity to >2 MelAgs. Regression of >1 tumor metastases were observed in seven of these patients. The overall immunity to MelAgs after DC vaccination is associated with clinical outcome (P = 0.015).

Dendritic cells: On the move from bench to bedside.

As dendritic cells increasingly become the adjuvant of choice in new approaches to cancer immunotherapy, a degree of protocol standardization is required to aid future large-scale clinical trials.

Sensing pathogens and tuning immune responses.

The immune system is capable of making qualitatively distinct responses against different microbial infections, and recent advances are starting to reveal how it manages this complex task. An integral component of the immune system is a network of cells known as dendritic cells (DCs), which sense different microbial stimuli and convey this information to lymphocytes. A better understanding of DC biology has allowed a model to be constructed in which the type of immune response to an infection is viewed as a function of several determinants, including the subpopulation of DCs, the nature of the microbe, microbe recognition receptors, and the cytokine microenvironment.

Modulating the immune response with dendritic cells and their growth factors.

Different subsets of dendritic cells (DCs) appear to play a role in determining the specific cytokines secreted by T helper (Th) cells. A model is proposed that links together factors such as the pathogen, microenvironment, DCs and T cells in a mechanism that results in a flexible determination of T-cell polarization.

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.

Identification of centerin: a novel human germinal center B cell-restricted serpin.

For naive B cells to mature in response to antigen triggering and become either plasma cells or memory B cells, a complex array of events takes place within germinal centers (GC) of secondary lymphoid organs. With the long-term objective of defining and characterizing molecules that control the generation of GC, we have subtracted RNA messages derived from highly purified B cells at the follicular mantle stage of differentiation from GC B cells. Using this approach, we have identified a novel molecule, centerin, belonging to the family of serine-protease inhibitors or serpins. Transcription of centerin is highly restricted to GC B cells and their malignant counterparts, Burkitt's lymphoma lines. The putative centerin protein shares the highest sequence identity with thyroxine-binding globulin and possesses arginine/serine at its P1/P1' active site, suggesting that it interacts with a trypsin-like protease(s). In addition, several other sequence features of centerin also indicate that it serves as a bonafide protease inhibitor. Finally, we demonstrate differentially up-regulated transcription of this novel gene by resting, naive B cells stimulated in vitro via CD40 signaling, while Staphylococcus aureus Cowan strain-mediated B cell activation fails to generate this reponse. Because CD40 signaling is required for naive B cells to enter the GC reaction and for GC B cells to survive, it is likely that centerin plays a role in the development and/or sustaining of GC.

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.

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.

Immunobiology of dendritic cells.

Dendritic cells (DCs) are antigen-presenting cells with a unique ability to induce primary immune responses. DCs capture and transfer information from the outside world to the cells of the adaptive immune system. DCs are not only critical for the induction of primary immune responses, but may also be important for the induction of immunological tolerance, as well as for the regulation of the type of T cell-mediated immune response. Although our understanding of DC biology is still in its infancy, we are now beginning to use DC-based immunotherapy protocols to elicit immunity against cancer and infectious diseases.