Nafamostat reduces systemic inflammation in TLR7-mediated virus-like illness.

BACKGROUND: The serine protease inhibitor nafamostat has been proposed as a treatment for COVID-19, by inhibiting TMPRSS2-mediated viral cell entry. Nafamostat has been shown to have other, immunomodulatory effects, which may be beneficial for treatment, however animal models of ssRNA virus infection are lacking. In this study, we examined the potential of the dual TLR7/8 agonist R848 to mimic the host response to an ssRNA virus infection and the associated behavioural response. In addition, we evaluated the anti-inflammatory effects of nafamostat in this model. METHODS: CD-1 mice received an intraperitoneal injection of R848 (200 µg, prepared in DMSO, diluted 1:10 in saline) or diluted DMSO alone, and an intravenous injection of either nafamostat (100 µL, 3 mg/kg in 5% dextrose) or 5% dextrose alone. Sickness behaviour was determined by temperature, food intake, sucrose preference test, open field and forced swim test. Blood and fresh liver, lung and brain were collected 6 h post-challenge to measure markers of peripheral and central inflammation by blood analysis, immunohistochemistry and qPCR. RESULTS: R848 induced a robust inflammatory response, as evidenced by increased expression of TNF, IFN-γ, CXCL1 and CXCL10 in the liver, lung and brain, as well as a sickness behaviour phenotype. Exogenous administration of nafamostat suppressed the hepatic inflammatory response, significantly reducing TNF and IFN-γ expression, but had no effect on lung or brain cytokine production. R848 administration depleted circulating leukocytes, which was restored by nafamostat treatment. CONCLUSIONS: Our data indicate that R848 administration provides a useful model of ssRNA virus infection, which induces inflammation in the periphery and CNS, and virus infection-like illness. In turn, we show that nafamostat has a systemic anti-inflammatory effect in the presence of the TLR7/8 agonist. Therefore, the results indicate that nafamostat has anti-inflammatory actions, beyond its ability to inhibit TMPRSS2, that might potentiate its anti-viral actions in pathologies such as COVID-19.

Sex differences in innate anti-viral immune responses to respiratory viruses and in their clinical outcomes in a birth cohort study.

The mechanisms explaining excess morbidity and mortality in respiratory infections among males are poorly understood. Innate immune responses are critical in protection against respiratory virus infections. We hypothesised that innate immune responses to respiratory viruses may be deficient in males. We stimulated peripheral blood mononuclear cells from 345 participants at age 16 years in a population-based birth cohort with three live respiratory viruses (rhinoviruses A16 and A1, and respiratory syncytial virus) and two viral mimics (R848 and CpG-A, to mimic responses to SARS-CoV-2) and investigated sex differences in interferon (IFN) responses. IFN-α responses to all viruses and stimuli were 1.34-2.06-fold lower in males than females (P = 0.018 - < 0.001). IFN-ß, IFN-γ and IFN-induced chemokines were also deficient in males across all stimuli/viruses. Healthcare records revealed 12.1% of males and 6.6% of females were hospitalized with respiratory infections in infancy (P = 0.017). In conclusion, impaired innate anti-viral immunity in males likely results in high male morbidity and mortality from respiratory virus infections.

Induction of trained immunity by influenza vaccination - impact on COVID-19.

Non-specific protective effects of certain vaccines have been reported, and long-term boosting of innate immunity, termed trained immunity, has been proposed as one of the mechanisms mediating these effects. Several epidemiological studies suggested cross-protection between influenza vaccination and COVID-19. In a large academic Dutch hospital, we found that SARS-CoV-2 infection was less common among employees who had received a previous influenza vaccination: relative risk reductions of 37% and 49% were observed following influenza vaccination during the first and second COVID-19 waves, respectively. The quadrivalent inactivated influenza vaccine induced a trained immunity program that boosted innate immune responses against various viral stimuli and fine-tuned the anti-SARS-CoV-2 response, which may result in better protection against COVID-19. Influenza vaccination led to transcriptional reprogramming of monocytes and reduced systemic inflammation. These epidemiological and immunological data argue for potential benefits of influenza vaccination against COVID-19, and future randomized trials are warranted to test this possibility.

Bovine innate immune phenotyping via a standardized whole blood stimulation assay.

Cattle vary in their susceptibility to infection and immunopathology, but our ability to measure and longitudinally profile immune response variation is limited by the lack of standardized immune phenotyping assays for high-throughput analysis. Here we report longitudinal innate immune response profiles in cattle using a low-blood volume, whole blood stimulation system-the ImmunoChek (IChek) assay. By minimizing cell manipulation, our standardized system minimizes the potential for artefactual results and enables repeatable temporal comparative analysis in cattle. IChek successfully captured biological variation in innate cytokine (IL-1ß and IL-6) and chemokine (IL-8) responses to 24-hr stimulation with either Gram-negative (LPS), Gram-positive (PamCSK4) bacterial or viral (R848) pathogen-associated molecular patterns (PAMPs) across a 4-month time window. Significant and repeatable patterns of inter-individual variation in cytokine and chemokine responses, as well as consistent high innate immune responder individuals were identified at both baseline and induced levels. Correlation coefficients between immune response read-outs (IL-1ß, IL-6 and IL-8) varied according to PAMP. Strong significant positive correlations were observed between circulating monocytes and IL-6 levels for null and induced responses (0.49-0.61) and between neutrophils and cytokine responses to R848 (0.38-0.47). The standardized assay facilitates high-throughput bovine innate immune response profiling to identify phenotypes associated with disease susceptibility and responses to vaccination.

Intratumoral immunotherapy using platelet-cloaked nanoparticles enhances antitumor immunity in solid tumors.

Intratumoral immunotherapy is an emerging modality for the treatment of solid tumors. Toll-like receptor (TLR) agonists have shown promise for eliciting immune responses, but systemic administration often results in the development of adverse side effects. Herein, we investigate whether localized delivery of the TLR agonist, resiquimod (R848), via platelet membrane-coated nanoparticles (PNP-R848) elicits antitumor responses. The membrane coating provides a means of enhancing interactions with the tumor microenvironment, thereby maximizing the activity of R848. Intratumoral administration of PNP-R848 strongly enhances local immune activation and leads to complete tumor regression in a colorectal tumor model, while providing protection against repeated tumor re-challenges. Moreover, treatment of an aggressive breast cancer model with intratumoral PNP-R848 delays tumor growth and inhibits lung metastasis. Our findings highlight the promise of locally delivering immunostimulatory payloads using biomimetic nanocarriers, which possess advantages such as enhanced biocompatibility and natural targeting affinities.

Sterilizing Immunity against SARS-CoV-2 Infection in Mice by a Single-Shot and Lipid Amphiphile Imidazoquinoline TLR7/8 Agonist-Adjuvanted Recombinant Spike Protein Vaccine*.

The search for vaccines that protect from severe morbidity and mortality because of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19) is a race against the clock and the virus. Here we describe an amphiphilic imidazoquinoline (IMDQ-PEG-CHOL) TLR7/8 adjuvant, consisting of an imidazoquinoline conjugated to the chain end of a cholesterol-poly(ethylene glycol) macromolecular amphiphile. It is water-soluble and exhibits massive translocation to lymph nodes upon local administration through binding to albumin, affording localized innate immune activation and reduction in systemic inflammation. The adjuvanticity of IMDQ-PEG-CHOL was validated in a licensed vaccine setting (quadrivalent influenza vaccine) and an experimental trimeric recombinant SARS-CoV-2 spike protein vaccine, showing robust IgG2a and IgG1 antibody titers in mice that could neutralize viral infection in vitro and in vivo in a mouse model.

A patient with melanoma that became sensitized to immunotherapy after treatment with a CDK4/6 inhibitor.

Background: Despite the profound effect that checkpoint inhibitors and BRAF/MEK inhibitors have had on survival in patients with metastatic melanoma, treatment options remain limited for those who demonstrate poor response or develop resistance to these modalities. The prospect of tumor sensitization to these treatments is therefore an attractive one. Results: We describe the case of a patient who developed a sustained response to trametinib and pembrolizumab, despite prior resistance to both these therapies, after receiving treatment with a CDK4/6 inhibitor. Discussion: We further outline the preclinical data supporting a possible role for the use of CDK4/6 inhibitors in tumor sensitization to immunotherapy.

Novel Lipidated Imidazoquinoline TLR7/8 Adjuvants Elicit Influenza-Specific Th1 Immune Responses and Protect Against Heterologous H3N2 Influenza Challenge in Mice.

Most licensed seasonal influenza vaccines are non-adjuvanted and rely primarily on vaccine-induced antibody titers for protection. As such, seasonal antigenic drift and suboptimal vaccine strain selection often results in reduced vaccine efficacy. Further, seasonal H3N2 influenza vaccines demonstrate poor efficacy compared to H1N1 and influenza type B vaccines. New vaccines, adjuvants, or delivery technologies that can induce broader or cross-seasonal protection against drifted influenza virus strains, likely through induction of protective T cell responses, are urgently needed. Here, we report novel lipidated TLR7/8 ligands that act as strong adjuvants to promote influenza-virus specific Th1-and Th17-polarized T cell responses and humoral responses in mice with no observable toxicity. Further, the adjuvanted influenza vaccine provided protection against a heterologous H3N2 influenza challenge in mice. These responses were further enhanced when combined with a synthetic TLR4 ligand adjuvant. Despite differences between human and mouse TLR7/8, these novel lipidated imidazoquinolines induced the production of cytokines required to polarize a Th1 and Th17 immune response in human PBMCs providing additional support for further development of these compounds as novel adjuvants for the induction of broad supra-seasonal protection from influenza virus.

Vaccination with whole-cell killed or recombinant leishmanial protein and toll-like receptor agonists against Leishmania tropica in BALB/c mice.

One strategy to control leishmaniasis is vaccination with potent antigens alongside suitable adjuvants. The use of toll-like receptor (TLR) agonists as adjuvants is a promising approach in Leishmania vaccine research. Leishmania (L.) tropica is among the less-investigated Leishmania species and a causative agent of cutaneous and sometimes visceral leishmaniasis with no approved vaccine against it. In the present study, we assessed the adjuvant effects of a TLR4 agonist, monophosphoryl lipid A (MPL) and a TLR7/8 agonist, R848 beside two different types of Leishmania vaccine candidates; namely, whole-cell soluble L. tropica antigen (SLA) and recombinant L. tropica stress-inducible protein-1 (LtSTI1). BALB/c mice were vaccinated three times by the antigens (SLA or LtSTI1) with MPL or R848 and then were challenged by L. tropica. Delayed-type hypersensitivity (DTH), parasite load, disease progression and cytokines (IL-10 and IFN-γ) responses were assessed. In general compared to SLA, application of LtSTI1 resulted in higher DTH, higher IFN-γ response and lower lymph node parasite load. Also compared to R848, MPL as an adjuvant resulted in higher DTH and lower lymph node parasite load. Although, no outstanding ability for SLA and R848 in evoking immune responses of BALB/c mice against L. tropica infection could be observed, our data suggest that LtSTI1 and MPL have a better potential to control L. tropica infection and could be pursued for the development of effective vaccination strategies.

The choice of linker for conjugating R848 to inactivated influenza virus determines the stimulatory capacity for innate immune cells.

Inactivated influenza vaccines are not approved for use in infants less than 6 months of age due to poor immunogenicity in that population. While the live attenuated influenza vaccine has the potential to be more immunogenic, it is not an option for infants and other vulnerable populations, including the elderly and immunocompromised individuals due to safety concerns. In an effort to improve the immunogenicity of the inactivated vaccine for use in vulnerable populations, we have used an approach of chemically crosslinking the Toll-like receptor (TLR) 7/8 agonist R848 directly to virus particles. We have reported previously that an R848-conjugated, inactivated vaccine is more effective at inducing adaptive immune responses and protecting against lung pathology in influenza challenged neonatal African green monkeys than is the unmodified counterpart. In the current study, we describe a second generation vaccine that utilizes an amide-sulfhydryl crosslinker with different spacer chemistry and length to couple R848 to virions. The new vaccine has significantly enhanced immunostimulatory activity for murine macrophages and importantly for monocyte derived human dendritic cells. Demonstration of the significant differences in stimulatory activity afforded by modest changes in linker impacts our fundamental view of the design of TLR agonist-antigen vaccines.

Functional, signalling and transcriptional differences of three distinct type I IFNs in a perciform fish, the mandarin fish Siniperca chuatsi.

Teleost fish are unique in having type I and type II interferons (IFNs) only, and the type I IFNs are classified into Group one and Group two based on the presence of two or four cysteines respectively, and are further classified into seven subgroups. In the present study, three distinct type I IFNs, IFNc, IFNd and IFNh, have been identified in the genome sequences of a perciform fish, the mandarin fish Siniperca chuatsi. These IFNs are induced following the stimulation of Polyinosinic polycytidylic acid (poly(I:C)) and Resiquimod (R848) either in vivo or in vitro. But, the infectious spleen and kidney necrosis virus (ISKNV) infection caused a delayed response of IFNs, which may be resulted from the viral inhibition of type I IFN production and related signalling. The three receptor subunits, cytokine receptor family B 1 (CRFB1), CRFB2 and CRFB5 are also expressed in a similar manner as observed for the IFNs, and IFNc, IFNd and IFNh use preferentially the receptor complex, CRFB2 and CRFB5, CRFB1 and CRFB5, CRFB1 and CRFB5 respectively for their effective signalling in the induction of IFN-stimulated genes (ISGs). Moreover, the IFNs are able to induce their own expression, and also the IRF3 and IRF7 expression, leading to the amplification of IFN cascade. It is further revealed that these three IFNs are transcribed differently by IRF7 and IRF3. The composition, function, signalling and transcription of type I IFNs have been investigated in detail in a teleost fish.

An R848 adjuvanted influenza vaccine promotes early activation of B cells in the draining lymph nodes of non-human primate neonates.

Impaired immune responsiveness is a significant barrier to vaccination of neonates. By way of example, the low seroconversion observed following influenza vaccination has led to restriction of its use to infants over 6 months of age, leaving younger infants vulnerable to infection. Our previous studies using a non-human primate neonate model demonstrated that the immune response elicited following vaccination with inactivated influenza virus could be robustly increased by inclusion of the Toll-like receptor agonist flagellin or R848, either delivered individually or in combination. When delivered individually, R848 was found to be the more effective of the two. To gain insights into the mechanism through which these adjuvants functioned in vivo, we assessed the initiation of the immune response, i.e. at 24 hr, in the draining lymph node of neonate non-human primates. Significant up-regulation of co-stimulatory molecules on dendritic cells could be detected, but only when both adjuvants were present. In contrast, R848 alone could increase the number of cells in the lymph node, presumably through enhanced recruitment, as well as B-cell activation at this early time-point. These changes were not observed with flagellin and the dual adjuvanted vaccine did not promote increases beyond those observed with R848 alone. In vitro studies showed that R848 could promote B-cell activation, supporting a model wherein a direct effect on neonate B-cell activation is an important component of the in vivo potency of R848 in neonates.

Evaluation of the adjuvant effect of agonists of toll-like receptor 4 and 7/8 in a vaccine against leishmaniasis in BALB/c mice.

There is no effective vaccine against human leishmaniasis. Achieving successful vaccines seems to need powerful adjuvants. Separate or combined use of toll like receptor (TLR) agonists as adjuvant is a promising approach in Leishmania vaccine research. In present study, we evaluated adjuvant effect of separate or combined use of a TLR7/8 agonist, R848 and a TLR4 agonist, monophosphoryl lipid A (MPL) beside soluble Leishmania antigen (SLA) in BALB/c mice. Mice were vaccinated three times by SLA with separate or combined TLR7/8 and TLR4 agonists and were then challenged by Leishmania major. Delay type hypersensitivity, lesion development, parasite load, and cytokines (interferon gamma, and interleukin-10) response were assessed. Results showed: 1) MPL can slightly assist SLA in parasite load reduction, but it is not able to increase SLA ability in evoking DTH and cytokine responses or decreasing lesion diameter. 2) R848 does not affect the DTH response and parasite load of mice vaccinated with SLA, but it decreases/inhibits cytokine responses induced by SLA, leading to increase lesion diameter. 3) MPL neutralized inhibitory effect of R848. In overall, these data emphasize that MPL slightly assists SLA to make a more potent vaccine, but R848 is not a good adjuvant to induce T cell-dependent immune response in BALB/c mice, and therefore combination of these TLR agonists in the current formulation, is not recommended for making a more powerful adjuvant.

Systematic Investigation of Multi-TLR Sensing Identifies Regulators of Sustained Gene Activation in Macrophages.

A typical pathogen presents a combination of Toll-like receptor (TLR) ligands during infection. Although individual TLR pathways have been well characterized, the nature of this "combinatorial code" in pathogen sensing remains unclear. Here, we conducted a comprehensive transcriptomic analysis of primary macrophages stimulated with all possible pairwise combinations of four different TLR ligands to understand the requirements, kinetics, and outcome of combined pathway engagement. We find that signal integration between TLR pathways leads to non-additive responses for a subset of immune mediators with sustained expression (>6 hr) properties and T cell polarizing function. To identify the underlying regulators, we conducted a focused RNAi screen and identified four genes-Helz2, Phf11d, Sertad3, and Zscan12-which preferentially affect the late phase response of TLR-induced immune effector expression. This study reveals key molecular details of how contemporaneous signaling through multiple TLRs, as would often be the case with pathogen infection, produce biological outcomes distinct from the single ligands typically used to characterize TLR pathways.

Intravaginal immunisation using a novel antigen-releasing ring device elicits robust vaccine antigen-specific systemic and mucosal humoral immune responses.

The generation of effective levels of antigen-specific immunity at the mucosal sites of pathogen entry is a key goal for vaccinologists. We explored topical vaginal application as an approach to initiate local antigen-specific immunity, enhance previously existing systemic immunity or re-target responses to the mucosae. To deliver a protein vaccine formulation to the vaginal mucosal surface, we used a novel vaginal ring device comprising a silicone elastomer body into which three freeze-dried, rod-shaped, hydroxypropylmethylcellulose inserts were incorporated. Each rod contained recombinant HIV-1 CN54gp140 protein (167µg)±R848 (167µg) adjuvant. The inserts were loaded into cavities within each ring such that only the ends of the inserts were initially exposed. Sheep received a prime-boost vaccination regime comprising intramuscular injection of 100µg CN54gp140+200µg R848 followed by three successive ring applications of one week duration and separated by one month intervals. Other sheep received only the ring devices without intramuscular priming. Serum and vaginal mucosal fluids were sampled every two weeks and analysed by CN54gp140 ELISA and antigen-specific B cells were measured by flow cytometry at necropsy. Vaccine antigen-specific serum antibody responses were detected in both the intramuscularly-primed and vaginal mucosally-primed groups. Those animals that received only vaginal vaccinations had identical IgG but superior IgA responses. Analysis revealed that all animals exhibited mucosal antigen-specific IgG and IgA with the IgA responses 30-fold greater than systemic levels. Importantly, very high numbers of antigen-specific B cells were detected in local genital draining lymph nodes. We have elicited local genital antigen-specific immune responses after topical application of an adjuvanted antigen formulation within a novel vaginal ring vaccine release device. This regimen and delivery method elicited high levels of antigen-specific mucosal IgA and large numbers of local antigen-reactive B cells, both likely essential for effective mucosal protection.

An immunostimulatory dual-functional nanocarrier that improves cancer immunochemotherapy.

Immunochemotherapy combines a chemotherapeutic agent with an immune-modulating agent and represents an attractive approach to improve cancer therapy. However, the success of immunochemotherapy is hampered by the lack of a strategy to effectively co-deliver the two therapeutics to the tumours. Here we report the development of a dual-functional, immunostimulatory nanomicellar carrier that is based on a prodrug conjugate of PEG with NLG919, an indoleamine 2,3-dioxygenase (IDO) inhibitor currently used for reversing tumour immune suppression. An Fmoc group, an effective drug-interactive motif, is also introduced into the carrier to improve the drug loading capacity and formulation stability. We show that PEG2k-Fmoc-NLG alone is effective in enhancing T-cell immune responses and exhibits significant antitumour activity in vivo. More importantly, systemic delivery of paclitaxel (PTX) using the PEG2k-Fmoc-NLG nanocarrier leads to a significantly improved antitumour response in both breast cancer and melanoma mouse models.

Adsorption of a synthetic TLR7/8 ligand to aluminum oxyhydroxide for enhanced vaccine adjuvant activity: A formulation approach.

For nearly a century, aluminum salts have been the most widely used vaccine adjuvant formulation, and have thus established a history of safety and efficacy. Nevertheless, for extremely challenging disease targets such as tuberculosis or HIV, the adjuvant activity of aluminum salts may not be potent enough to achieve protective efficacy. Adsorption of TLR ligands to aluminum salts facilitates enhanced adjuvant activity, such as in the human papilloma virus vaccine Cervarix®. However, some TLR ligands such as TLR7/8 agonist imidazoquinolines do not efficiently adsorb to aluminum salts. The present report describes a formulation approach to solving this challenge by developing a lipid-based nanosuspension of a synthetic TLR7/8 ligand (3M-052) that facilitates adsorption to aluminum oxyhydroxide via the structural properties of the helper lipid employed. In immunized mice, the aluminum oxyhydroxide-adsorbed formulation of 3M-052 enhanced antibody and TH1-type cellular immune responses to vaccine antigens for tuberculosis and HIV.

Butyrophilin 3A/CD277-Dependent Activation of Human γδ T Cells: Accessory Cell Capacity of Distinct Leukocyte Populations.

Human Vγ9Vδ2 T cells recognize in a butyrophilin 3A/CD277-dependent way microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) or endogenous pyrophosphates (isopentenyl pyrophosphate [IPP]). Nitrogen-bisphosphonates such as zoledronic acid (ZOL) trigger selective γδ T cell activation because they stimulate IPP production in monocytes by inhibiting the mevalonate pathway downstream of IPP synthesis. We performed a comparative analysis of the capacity of purified monocytes, neutrophils, and CD4 T cells to serve as accessory cells for Vγ9Vδ2 T cell activation in response to three selective but mechanistically distinct stimuli (ZOL, HMBPP, agonistic anti-CD277 mAb). Only monocytes supported γδ T cell expansion in response to all three stimuli, whereas both neutrophils and CD4 T cells presented HMBPP but failed to induce γδ T cell expansion in the presence of ZOL or anti-CD277 mAb. Preincubation of accessory cells with the respective stimuli revealed potent γδ T cell-stimulating activity of ZOL- or anti-CD277 mAb-pretreated monocytes, but not neutrophils. In comparison with monocytes, ZOL-pretreated neutrophils produced little, if any, IPP and expressed much lower levels of farnesyl pyrophosphate synthase. Exogenous IL-18 enhanced the γδ T cell expansion with all three stimuli, remarkably also in response to CD4 T cells and neutrophils preincubated with anti-CD277 mAb or HMBPP. Our study uncovers unexpected differences between monocytes and neutrophils in their accessory function for human γδ T cells and underscores the important role of IL-18 in driving γδ T cell expansion. These results may have implications for the design of γδ T cell-based immunotherapeutic strategies.

Maturation and cytokine production potential of dendritic cells isolated from rheumatoid arthritis patients peripheral blood and induced in vitro.

BACKGROUND: Since dendritic cells (DC) are involved in the development of autoimmune inflammation, researchers consider DC both as target cells for specific therapy of rheumatoid arthritis (RA) and as candidate cells for the development of cell-based methods to treat autoimmune diseases. The development of treatment strategies requires comprehensive research into the quantitative and qualitative characteristics of DC subtypes both ex vivo from RA patients and in vitro, to determine the possibility of inducing functionally mature DC in RA. OBJECTIVE: To study the phenotypic and functional properties of myeloid (mDC) and plasmacytoid (pDC) DC isolated from the peripheral blood of patients with RA and induced in vitro. MATERIALS AND METHODS: Blood samples were obtained from RA patients and healthy donors. Immature DC in the whole blood and in vitro induced DC were characterized by the positive expression of CD80, CD83, CCR7, IL-10, IL-4, IL-12 and IFN-α. R848 and lipopolysaccharide were used to determine DC maturation ability. From PBMCs of RA patients and health donors DCs with myeloid (imDC) and plasmacytoid (ipDC) phenotype were induced. RESULTS: The relative count of mDC in the peripheral blood between studied groups did not differ. pDC count was significantly lower for RA patients. DC from RA patients were characterized by low expression levels of CD80 and CD83 on both populations cells and high expression of CCR7 only on pDC. An increase in pDC producing IL-12 and IFN-α and a decrease in mDC and pDC producing IL-4 and IL-10 were shown in RA. imDC and ipDC obtained from RA patients according to their phenotype and cytokine profile did not differ from those obtained from healthy donors. CONCLUSIONS: There is an imbalance between subpopulations of DC in the peripheral blood of RA patients. DC of RA patients are less mature. The data suggest the involvement of DC in RA pathogenesis and confirm DC participation in balance shift towards Th1-type immune responses. At the same time, in vitro induced RA DC are phenotypically and functionally competent.