TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2.

Tumor necrosis factor (TNF)-α is a pleiotropic cytokine implicated in the etiology of several autoimmune diseases, including rheumatoid arthritis (RA). TNF-α regulates diverse effector functions through the activation of TNF-α receptor (TNFR)1 and TNFR2. Although the detrimental role of this cytokine has been addressed in distinct disease settings, the effects of TNF-α on cytokine production by isolated CD4+ T helper type 1 (Th1) and Th17 cells, two T cell subpopulations that contribute to the pathogenesis of RA, have not been completely elucidated. Here, we show that TNF-α promotes a reduction and expansion in the frequency of both T cell subsets producing IFN-γ and IL-17, respectively. Selective blockade of TNFR1 or TNFR2 on Th1 and Th17 cells revealed that TNFR2 mediates the decrease in IFN-γ production, while signaling through both receptors augments IL-17 production. We also demonstrate that Th1, but not Th17 cells from RA patients present lower levels of TNFR1 compared to healthy controls, whereas TNFR2 expression on both T cell types is similar between patients and controls. Since TNF-α receptors levels in RA patients are not significantly changed by the therapeutic blockade of TNF-α, we propose that targeting TNFR2 may represent an alternative strategy to normalize the levels of key cytokines that contribute to RA pathogenesis.

Transcription factor engineering in CHO cells for recombinant protein production.

The continuous increase of approved biopharmaceutical products drives the development of more efficient recombinant protein expression systems. Chinese hamster ovary (CHO) cells are the mainstay for this purpose but have some drawbacks, such as low levels of expression. Several strategies have been applied to increase the productivity of CHO cells with different outcomes. Transcription factor (TF) engineering has emerged as an interesting and successful approach, as these proteins can act as master regulators; the expression and function of a TF can be controlled by small molecules, and it is possible to design tailored TFs and promoters with desired features. To date, the majority of studies have focused on the use of TFs with growth, metabolic, cell cycle or endoplasmic reticulum functions, although there is a trend to develop new, synthetic TFs. Moreover, new synthetic biological approaches are showing promising advances for the development of specific TFs, even with tailored ligand sensitivity. In this article, we summarize the strategies to increase recombinant protein expression by modulating and designing TFs and with advancements in synthetic biology. We also illustrate how this class of proteins can be used to develop more robust expression systems.

Neuropilin-1+ regulatory T cells promote skin allograft survival and modulate effector CD4+ T cells phenotypic signature.

During allograft rejection, several immune cell types, including dendritic cells, CD4(+) and CD8(+) T cells among others, recirculate between the graft and the nearest draining lymph node, resulting in immunity against the 'foreign' tissue. Regulatory CD4(+) T cells are critical for controlling the magnitude of the immune response and may act to promote or maintain tolerance. They are characterized by the expression of CD25 and Foxp3, and more recently, Neuropilin-1 (Nrp1). The role of these suppressor cells during allograft rejection is not well understood. Our work shows that during graft rejection, there is an increase in the frequency of total CD4(+) T cells expressing Nrp1, but the expression of this molecule is downregulated in the regulatory CD4(+) T-cell compartment. Interestingly, the expression of the transcription factor Eos, which renders cell function stability, is also reduced. In adoptive transfer experiments, we observed that during allograft rejection: (i) natural regulatory CD4(+) T cells maintain high levels of Nrp1 expression, (ii) effector CD4(+) T cells (Nrp1(-)) become Nrp1(+)Eos(+) and (iii) the transfer of regulatory CD4(+) T cells (Nrp1(+)) can promote allograft survival, and also enhance the gain of Nrp1 and Eos on T-effector cells. Together, these data suggest that rejection occurs, at least in part, through the loss of Nrp1 expression on regulatory CD4(+) T cells, their stability or both. Additionally, the transfer of regulatory CD4(+) T cells (based on Nrp1 expression) permits the acceptance of the allograft, placing Nrp1 as a new target for immune therapy.

A short protocol using dexamethasone and monophosphoryl lipid A generates tolerogenic dendritic cells that display a potent migratory capacity to lymphoid chemokines.

BACKGROUND: Generation of tolerogenic dendritic cells (TolDCs) for therapy is challenging due to its implications for the design of protocols suitable for clinical applications, which means not only using safe products, but also working at defining specific biomarkers for TolDCs identification, developing shorter DCs differentiation methods and obtaining TolDCs with a stable phenotype. We describe here, a short-term protocol for TolDCs generation, which are characterized in terms of phenotypic markers, cytokines secretion profile, CD4+ T cell-stimulatory ability and migratory capacity. METHODS: TolDCs from healthy donors were generated by modulation with dexamethasone plus monophosphoryl lipid A (MPLA-tDCs). We performed an analysis of MPLA-tDCs in terms of yield, viability, morphology, phenotypic markers, cytokines secretion profile, stability, allogeneic and antigen-specific CD4+ T-cell stimulatory ability and migration capacity. RESULTS: After a 5-day culture, MPLA-tDCs displayed reduced expression of costimulatory and maturation molecules together to an anti-inflammatory cytokines secretion profile, being able to maintain these tolerogenic features even after the engagement of CD40 by its cognate ligand. In addition, MPLA-tDCs exhibited reduced capabilities to stimulate allogeneic and antigen-specific CD4+ T cell proliferation, and induced an anti-inflammatory cytokine secretion pattern. Among potential tolerogenic markers studied, only TLR-2 was highly expressed in MPLA-tDCs when compared to mature and immature DCs. Remarkable, like mature DCs, MPLA-tDCs displayed a high CCR7 and CXCR4 expression, both chemokine receptors involved in migration to secondary lymphoid organs, and even more, in an in vitro assay they exhibited a high migration response towards CCL19 and CXCL12. CONCLUSION: We describe a short-term protocol for TolDC generation, which confers them a stable phenotype and migratory capacity to lymphoid chemokines, essential features for TolDCs to be used as therapeutics for autoimmunity and prevention of graft rejection.

Weak CD4+ T-cell responses to citrullinated vimentin in rheumatoid arthritis patients carrying HLA-DR9 alleles.

Citrullinated vimentin (cVIM) is one of the antigens specifically targeted by anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) patients. The association between ACPA and certain HLA-DRB1 alleles, those coding for the shared epitope (SE), suggests that this response could be T-cell mediated. HLA-DR9 alleles, which do not code for the SE, have recently been associated with ACPA (+) RA. The objective of this work was to study CD4+ T cell responses to cVIM in RA patients and healthy controls carrying HLA-DR9 alleles. Fourteen RA patients and ten healthy controls previously genotyped for HLA-DRB1 were studied for the presence of serum anti-cVIM antibodies by Western blot and ELISA. Peripheral blood mononuclear cells were stimulated with native vimentin and cVIM, and CD4+ T cells proliferation was assessed by flow cytometry. Citrulline-specific CD4+ T cells proliferation was found not only in RA patients but also in healthy controls. Although most patients carrying HLA-DR9 alleles present anti-cVIM antibodies, HLA-DR9 alleles were associated with weaker cVIM-driven CD4+ T-cell responses among RA patients. These results suggest that HLA-DR9 alleles could exert a protective effect on the recognition of cVIM epitopes by CD4+ T cells. In this context, other citrullinated proteins may break T and B cell tolerance, with cVIM only acting as a cross-reactive target for ACPA.

Interleukin 10 decreases MICA expression on melanoma cell surface.

Natural-killer group 2, member D (NKG2D) binds to a variety of ligands, including the major histocompatibility complex (MHC) class I chain-related proteins (MIC) and UL16-binding proteins (ULBP). It is regarded as a co-activating receptor on NK cells, having an important role in the cell-mediated immune response to tumours. We studied the influence of interleukin (IL)-10 on the regulation of MIC and ULBP expression on melanoma cells, and its effect on the cytotoxic function of NK cells in vitro. Here, we show that, in the presence of IL-10, FMS mel and BL mel cell lines decreased MICA and ULBP2 surface expression, whereas MHC class I did not change substantially on the cell surface. MICA mRNA levels decreased in IL-10-treated FMS and IL-10-transduced BL cell lines. Interestingly, we observed that MICB surface expression and its mRNA levels increased upon IL-10 treatment in a melanoma cell line. These changes in NKG2D ligands surface expression patterns owing to IL-10 treatment resulted in an effect on lysis susceptibility mediated by lymphocyte-activated killer cells, as tumour cell lines that displayed a higher decrease of MICA on their surface had lower levels of lysis. In addition, expression of CD107a was downregulated on the surface of NK cells following stimulation with IL-10-treated FMS cells. Our results suggest a novel function for IL-10 in the modulation of NKG2D ligand expression and in the control of cytotoxicity mediated by NKG2D/NKG2D ligand axis.

Immunosuppressive Mechanisms of Regulatory B Cells.

Regulatory B cells (Bregs) is a term that encompasses all B cells that act to suppress immune responses. Bregs contribute to the maintenance of tolerance, limiting ongoing immune responses and reestablishing immune homeostasis. The important role of Bregs in restraining the pathology associated with exacerbated inflammatory responses in autoimmunity and graft rejection has been consistently demonstrated, while more recent studies have suggested a role for this population in other immune-related conditions, such as infections, allergy, cancer, and chronic metabolic diseases. Initial studies identified IL-10 as the hallmark of Breg function; nevertheless, the past decade has seen the discovery of other molecules utilized by human and murine B cells to regulate immune responses. This new arsenal includes other anti-inflammatory cytokines such IL-35 and TGF-ß, as well as cell surface proteins like CD1d and PD-L1. In this review, we examine the main suppressive mechanisms employed by these novel Breg populations. We also discuss recent evidence that helps to unravel previously unknown aspects of the phenotype, development, activation, and function of IL-10-producing Bregs, incorporating an overview on those questions that remain obscure.

Levels of interferon-gamma and transcription factor T-bet in progressive periodontal lesions in patients with chronic periodontitis.

BACKGROUND: Periodontitis is an infection with an episodic pattern of tissue-support destruction. During the generation of a primary CD4(+) T helper 1 (Th1) response, interferon-gamma (IFN-gamma) acts as a positive regulator by selectively inducing Th1 differentiation through increased transcription of T-bet. The aims of this work were to determine IFN-gamma levels in samples of gingival crevicular fluid (GCF) and to determine IFN-gamma and transcription factor T-bet expression in gingival tissue from patients undergoing the progression of chronic periodontitis. METHODS: One hundred six patients with moderate or advanced chronic periodontitis were selected. Periodontitis was characterized by at least six sites with probing depth >or=5 mm, clinical attachment loss >or=3 mm, and radiographic bone loss. Periodontitis progression was determined by the tolerance method. GCF was collected using a paper strip, and enzyme-linked immunosorbent assay was performed to determine the total amount of IFN-gamma. Gingival biopsies were obtained from patients for real-time reverse transcription-polymerase chain reaction to determine IFN-gamma and T-bet expression. Statistical analysis was performed using statistical software. Data were expressed as subject means +/- SD. The chi(2) and Student t tests were used. RESULTS: The total amount and concentration of cytokine IFN-gamma were significantly higher in active sites than in inactive sites (99.90 versus 68.90 pg; P = 0.03; 106.62 pg/mg versus 75.64 pg/mg, P = 0.04, respectively). Active sites showed a significantly lower Delta cycle threshold (Ct) of IFN-gamma than inactive sites (P = 0.04), whereas the expression of transcription factor T-bet was increased 1.42-fold in active sites compared to inactive sites. CONCLUSION: The total amount and concentration of cytokine IFN-gamma in GCF samples and transcription factor T-bet expression were increased in progressive periodontal lesions in patients with chronic periodontitis.

Humanized Mouse Models of Rheumatoid Arthritis for Studies on Immunopathogenesis and Preclinical Testing of Cell-Based Therapies.

Rodent models of rheumatoid arthritis (RA) have been used over decades to study the immunopathogenesis of the disease and to explore intervention strategies. Nevertheless, mouse models of RA reach their limit when it comes to testing of new therapeutic approaches such as cell-based therapies. Differences between the human and the murine immune system make it difficult to draw reliable conclusions about the success of immunotherapies. To overcome this issue, humanized mouse models have been established that mimic components of the human immune system in mice. Two main strategies have been pursued for humanization: the introduction of human transgenes such as human leukocyte antigen molecules or specific T cell receptors, and the generation of mouse/human chimera by transferring human cells or tissues into immunodeficient mice. Recently, both approaches have been combined to achieve more sophisticated humanized models of autoimmune diseases. This review discusses limitations of conventional mouse models of RA-like disease and provides a closer look into studies in humanized mice exploring their usefulness and necessity as preclinical models for testing of cell-based therapies in autoimmune diseases such as RA.

Development of a new promoter to avoid the silencing of genes in the production of recombinant antibodies in chinese hamster ovary cells.

BACKGROUND: The production of recombinant proteins in mammalian cell lines is one of the most important areas in biopharmaceutical industry. Viral transcriptional promoters are widely used to express recombinant proteins in mammalian cell lines. However, these promoters are susceptible to silencing, thus limiting protein productivity. Some CpG islands can avoid the silencing of housekeeping genes; for that reason, they have been used to increase the production of recombinant genes in cells of animal origin. In this study, we evaluated the CpG island of the promoter region of the ß-actin gene of Cricetulus griseous (Chinese hamster), associated to the Cytomegalovirus (CMV) promoter, to increase recombinant antibodies production in Chinese Hamster Ovary (CHO) cells. RESULTS: We focused on the non-coding region of CpG island, which we called RegCG. RegCG behaved as a promoter, whose transcriptional activity was mainly commanded by the CAAT and CArG boxes of the proximal promoter. However, the transcription started mainly at the intronic region before the proximal transcription start site. While the CMV promoter was initially more powerful than RegCG, the latter promoter was more resistant to silencing than the CMV promoter in stable cell lines, and its activity was improved when combined with the CMV promoter. Thereby, the chimeric promoter was able to maintain the expression of recombinant antibodies in stable clones for 40 days at an average level 4 times higher than the CMV promoter. Finally, the chimeric promoter showed compatibility with a genetic amplification system by induction with methotrexate in cells deficient in the dihydrofolate reductase gene. CONCLUSIONS: We have generated an efficient synthetic hybrid transcription promoter through the combination of RegCG with CMV, which, in stable cell lines, shows greater activity than when both promoters are used separately. Our chimeric promoter is compatible with a genetic amplification system in CHO DG44 cells and makes possible the generation of stable cell lines with high production of recombinant antibodies. We propose that this promoter can be a good alternative for the generation of clones expressing high amount of recombinant proteins, essential for industrial applications.

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC.

The potential of tolerogenic dendritic cells (tolDCs) to shape immune responses and restore tolerance has turn them into a promising therapeutic tool for cellular therapies directed toward immune regulation in autoimmunity. Although the cellular mechanisms by which these cells can exert their regulatory function are well-known, the mechanisms driving their differentiation and function are still poorly known, and the variety of stimuli and protocols applied to differentiate DCs toward a tolerogenic phenotype makes it even more complex to underpin the molecular features involved in their function. Through transcriptional profiling analysis of monocyte-derived tolDCs modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), known as DM-DCs, we were able to identify MYC as one of the transcriptional regulators of several genes differentially expressed on DM-DCs compared to MPLA-matured DCs (M-DCs) and untreated/immature DCs (DCs) as revealed by Ingenuity Pathway Analysis (IPA) upstream regulators evaluation. Additionally, MYC was also amidst the most upregulated genes in DM-DCs, finding that was confirmed at a transcriptional as well as at a protein level. Blockade of transactivation of MYC target genes led to the downregulation of tolerance-related markers IDO1 and JAG1. MYC blockade also led to downregulation of PLZF and STAT3, transcription factors associated with immune regulation and inhibition of DC maturation, further supporting a role of MYC as an upstream regulator contributing to the regulatory phenotype of DM-DCs. On the other hand, we had previously shown that fatty acid oxidation, oxidative metabolism and zinc homeostasis are amongst the main biological functions represented in DM-DCs, and here we show that DM-DCs exhibit higher intracellular expression of ROS and Zinc compared to mature M-DCs and DCs. Taken together, these findings suggest that the regulatory profile of DM-DCs is partly shaped by the effect of the transcriptional regulation of tolerance-inducing genes by MYC and the modulation of oxidative metabolic processes and signaling mediators such as Zinc and ROS.

Characterization of progressive periodontal lesions in chronic periodontitis patients: levels of chemokines, cytokines, matrix metalloproteinase-13, periodontal pathogens and inflammatory cells.

BACKGROUND AND AIMS: Periodontitis is an infection with an episodic nature of tissue support destruction. The aim of this work was to determine the levels of chemokines, cytokines, matrix metalloproteinase-13, periodontal pathogens and inflammatory cells in periodontal sites characterized by active periodontal connective tissue destruction. MATERIAL AND METHOD: Fifty-six patients with moderate or advanced severity of chronic periodontitis were selected. Periodontitis was characterized by at least six sites with probing depth > or =5 mm, clinical attachment level > or =3 mm and radiographic bone loss. Periodontitis progression was determined by the tolerance method. Receptor activator for nuclear factor kappa B-ligand (RANK-L), monocyte chemoattractant protein-1 (MCP-1), tumour necrosis factor-alpha (TNF-alpha), IL-1beta, MMP-13, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsithia and inflammatory cells levels were determined. Statistical analysis was performed using the Stata 7.0 software. Data were expressed as mean+/-SD and paired samples t-test and chi(2) tests were used. RESULTS: Higher RANK-L, IL-1beta and MMP-13 activity levels were observed in active sites (p<0.05). The proportion of P. gingivalis, A. actinomycetemcomitans, T. forsythia and the number of CD4(+) T were higher in active than in inactive sites (p>0.05). CONCLUSION: The detection of periodontopathic bacteria, host matrix metalloproteinases and cytokines in periodontitis patients with lesions undergoing episodic attachment loss could partially explain the mechanisms associated with the destruction of the supporting tissues of the tooth.

Mild hypothermia upregulates myc and xbp1s expression and improves anti-TNFα production in CHO cells.

Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on myc and xbp1s expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant anti-TNFα and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of anti-TNFα. Under mild hypothermia myc and xbp1s expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of myc and xbp1s in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line.

TIM-1 defines a human regulatory B cell population that is altered in frequency and function in systemic sclerosis patients.

BACKGROUND: Systemic sclerosis (SSc) is a systemic autoimmune disease characterized by excessive production of extracellular matrix by fibroblasts on skin and internal organs. Although Th2 cells have been involved in fibroblast stimulation, hyperactivated B cells may also play an important role. Regulatory B cells (Bregs) are cells capable of inhibiting inflammatory responses and controlling autoimmune diseases. Although many Breg populations have in common the ability to produce high amounts of IL-10, a unique surface marker defining most human Bregs is lacking. It has been described in mice that T cell Ig and mucin domain protein 1 (TIM-1) is an inclusive marker for Bregs, and that TIM-1+ B cells are able to prevent the development of autoimmunity. The aim of this work was to evaluate TIM-1 as a marker for human IL-10+ Bregs, and to determine whether TIM-1+ B cells are defective in SSc patients. METHODS: SSc patients (n = 39) and 53 healthy subjects were recruited. TIM-1 and IL-10 expression was assessed in resting or activated peripheral blood CD19+ B cells by flow cytometry. The regulatory function of TIM-1+ or activated B cells from SSc patients and healthy subjects was assessed in autologous and allogenic co-cultures with CD4+ T cells, where T cell proliferation and IFN-γ, IL-17, TNF-α and IL-4 production by T cells was measured by flow cytometry. RESULTS: TIM-1 and IL-10 were preferentially expressed in transitional B cells, but were upregulated in naïve and memory B cells upon stimulation. The frequency of transitional TIM-1+ IL-10+ B cells was significantly decreased in SSc patients compared to healthy controls. In addition, activated B cells from SSc patients induced stronger allogenic Th1 and Th2 responses than activated B cells from healthy controls. Finally, TIM-1+ B cells, including transitional and non-transitional cells, exhibited a higher CD4+ T cell suppressive ability than TIM-1- B cells in healthy controls, but not in SSc patients. CONCLUSIONS: TIM-1 is a unique marker for the identification of a human IL-10+ Breg subpopulation which is partially superimposed with transitional B cells. Alterations in TIM-1+ B cells could contribute to the development of autoimmune diseases such as SSc.

Triatoma infestans Calreticulin: Gene Cloning and Expression of a Main Domain That Interacts with the Host Complement System.

Triatoma infestans is an important hematophagous vector of Chagas disease, a neglected chronic illness affecting approximately 6 million people in Latin America. Hematophagous insects possess several molecules in their saliva that counteract host defensive responses. Calreticulin (CRT), a multifunctional protein secreted in saliva, contributes to the feeding process in some insects. Human CRT (HuCRT) and Trypanosoma cruzi CRT (TcCRT) inhibit the classical pathway of complement activation, mainly by interacting through their central S domain with complement component C1. In previous studies, we have detected CRT in salivary gland extracts from T. infestans We have called this molecule TiCRT. Given that the S domain is responsible for C1 binding, we have tested its role in the classical pathway of complement activation in vertebrate blood. We have cloned and characterized the complete nucleotide sequence of CRT from T. infestans, and expressed its S domain. As expected, this S domain binds to human C1 and, as a consequence, it inhibits the classical pathway of complement, at its earliest stage of activation, namely the generation of C4b. Possibly, the presence of TiCRT in the salivary gland represents an evolutionary adaptation in hematophagous insects to control a potential activation of complement proteins, present in the massive blood meal that they ingest, with deleterious consequences at least on the anterior digestive tract of these insects.

STAT3 inhibition by STA21 increases cell surface expression of MICB and the release of soluble MICB by gastric adenocarcinoma cells.

NKG2D is an activating receptor expressed on NK cells that binds to a variety of ligands, including MICA and MICB. These cell surface glycoproteins are overexpressed under cellular transformation, thus playing an important role in cell-mediated immune response to tumors. STAT3 is a transcription factor that is constitutively active in cancer. It negatively regulates MICA expression on target cells, while its inhibition enhances NK cell cytotoxicity against tumors. In this work, we aimed to describe the effect of STAT3 signaling inhibition by STA21 on the regulation of MICB expression in gastric adenocarcinoma cells and its effect on the cytotoxic function of NK cells. Treatment of gastric adenocarcinoma cells with STA21 induced an increase in MICB expression and soluble MICB secretion, as well as a variable pattern on effector cell degranulation. Soluble MICB secretion by gastric adenocarcinoma cells was not affected by metalloprotease inhibition. We also observed that primary gastric adenocarcinoma tissue released soluble MICB into the extracellular milieu. Recombinant MICB induced a significant decrease in the levels of NKG2D receptor on effector NK and CD8+ T cells, which correlated with an impaired cytotoxic function. Altogether, our data provide evidence that STAT3 signaling pathway regulates MICB expression on gastric adenocarcinoma cells and that recombinant soluble MICB compromises the cytolytic activity of NK cells.

The role of regulatory T lymphocytes in the induced immune response mediated by biological vaccines.

Immunotherapy has become a novel therapeutic alternative for various kinds of tumours. Recently, we have finalized the first phase I clinical study in Chile for the treatment of advanced malignant melanoma, using dendritic cells (DCs) loaded with allogeneic melanoma cell lysate. This study included 20 patients and the obtained results, pioneer in Latin America, showed that DC-based immunotherapy is innocuous, even provided in combination with IL-2. In addition, immunological responses were detected in 50% of the treated patients, establishing a positive correlation between the delayed type hypersensitivity (DTH) reaction, which indicates induction of in vivo immunological memory, and patients surviving. Nevertheless, objective clinical responses in vaccinated patients are still insufficient. Only sporadic objective metastasis regressions have been registered and an important proportion of the treated patients did not respond, or their responses were weak. Several strategies have been described to be used by tumours to escape from the immune response. Actually, we have demonstrated that IL-10 inhibits antigen presentation in melanoma, reducing tumour sensitivity to melanoma-specific cytotoxic T lymphocytes (CTLs). Regulation of the immunological response by inhibitory cells could be another possible cause of clinical unresponsiveness. Lately, the existence of subpopulations of regulatory T lymphocytes (RTL) able to limit the immune response in a specific form has been established, specially inhibiting the proliferation and activity of CD4+ and CD8+ effector T lymphocytes. These cellular subpopulations, mostly CD4+/CD25+/Foxp3+ T lymphocytes (Treg) of thymic origin, or TR1 lymphocytes able to release IL-10, and tumour growth factor beta (TGF-beta) producing TH3 lymphocytes, would be accumulated in the body during tumour growth, inhibiting the immune response. In relation to RTL and cancer, evidence indicates that Treg cell numbers are increased in blood and other tissues in different types of cancer. Additionally, it has been demonstrated that in patients with refractory metastatic melanoma, the adoptive transference of anti-tumour CD8+ T lymphocytes after non-myeloablative chemotherapy was able to induce important tumour regressions that would be due to elimination of RTL populations. Additionally, chemotherapeutical drugs like decarbazine, besides their effect on tumour proliferation, also have an immunosuppressive effect on T lymphocyte populations, as well as on accumulated RTL. In this article, a novel strategy for the study of RTL is proposed, including potential therapeutic innovations, which is being pioneered in current clinical trials.

Tolerogenic dendritic cells for reprogramming of lymphocyte responses in autoimmune diseases.

Dendritic cells (DCs) control immune responses by driving potent inflammatory actions against external and internal threats while generating tolerance to self and harmless components. This duality and their potential to reprogram immune responses in an antigen-specific fashion have made them an interesting target for immunotherapeutic strategies to control autoimmune diseases. Several protocols have been described for in vitro generation of tolerogenic DCs (tolDCs) capable of modulating adaptive immune responses and restoring tolerance through different mechanisms that involve anergy, generation of regulatory lymphocyte populations, or deletion of potentially harmful inflammatory T cell subsets. Recently, the capacity of tolDCs to induce interleukin (IL-10)-secreting regulatory B cells has been demonstrated. In vitro assays and rodent models of autoimmune diseases provide insights to the molecular regulators and pathways enabling tolDCs to control immune responses. Here we review mechanisms through which tolDCs modulate adaptive immune responses, particularly focusing on their suitability for reprogramming autoreactive CD4+ effector T cells. Furthermore, we discuss recent findings establishing that tolDCs also modulate B cell populations and discuss clinical trials applying tolDCs to patients with autoimmune diseases.

Clinical significance of tumor expression of major histocompatibility complex class I-related chains A and B (MICA/B) in gastric cancer patients.

Gastric cancer (GC) is the third most common cause of cancer death worldwide. Natural killer cells play an important role in the immune defense against transformed cells. They express the activating receptor NKG2D, whose ligands belong to the MIC and ULBP/RAET family. Although it is well established that these ligands are generally expressed in tumors, the association between their expression in the tumor and gastric mucosa and clinical parameters and prognosis of GC remains to be addressed. In the present study, MICA and MICB expression was analyzed, by flow cytometry, in 23 and 20 pairs of gastric tumor and adjacent non-neoplasic gastric mucosa, respectively. Additionally, ligands expression in 13 tumors and 7 gastric mucosa samples from GC patients were evaluated by immunohistochemistry. The mRNA levels of MICA in 9 pairs of tumor and mucosa were determined by quantitative PCR. Data were associated with the clinicopathological characteristics and the patient outcome. MICA expression was observed in 57% of tumors (13/23) and 44% of mucosal samples (10/23), while MICB was detected in 50% of tumors (10/20) and 45% of mucosal tissues (9/20). At the protein level, ligand expression was significantly higher in the tumor than in the gastric mucosa. MICA mRNA levels were also increased in the tumor as compared to the mucosa. However, clinicopathological analysis indicated that, in patients with tumors >5 cm, the expression of MICA and MICB in the tumor did not differ from that of the mucosa, and tumors >5 cm showed significantly higher MICA and MICB expression than tumors ≤5 cm. Patients presenting tumors >5 cm that expressed MICA and MICB had substantially shorter survival than those with large tumors that did not express these ligands. Our results suggest that locally sustained expression of MICA and MICB in the tumor may contribute to the malignant progression of GC and that expression of these ligands predicts an unfavorable prognosis in GC patients presenting large tumors.

Gene Expression Profiling of Human Monocyte-derived Dendritic Cells - Searching for Molecular Regulators of Tolerogenicity.

The ability of dendritic cells (DCs) to initiate and modulate antigen-specific immune responses has made them attractive targets for immunotherapy. Since DC research in humans is limited by the scarcity of DC populations in the blood circulation, most of our knowledge about DC biology and function has been obtained in vitro from monocyte-derived DCs (moDCs), which can be readily generated in sufficient numbers and are able to differentiate into distinct functional subsets depending on the nature of stimulus. In particular, moDCs with tolerogenic properties (tolDCs) possess great therapeutic potential for the treatment of autoimmune diseases. Several protocols have been developed to generate tolDCs in vitro, able to reinstruct auto-reactive T cells and to promote regulatory cells. While ligands and soluble mediators, by which DCs shape immune responses, have been vastly studied, the intracellular pathways and transcriptional regulators that govern tolDC differentiation and function are poorly understood. Whole-genome microarrays and proteomics provide useful strategies to dissect the complex molecular processes that promote tolerogenicity. Only few attempts have been made to understand tolDC biology through a global view on "omics" profiles. So far, the identification of a common regulator of tolerogenicity has been hampered by the fact that each protocol, used for tolDC generation, targets distinct signaling pathways. Here, we review the progress in understanding the transcriptional regulation of moDC differentiation, with a special focus on tolDCs, and highlight candidate molecules that might be associated with DC tolerogenicity.