Expression of Homing Receptors in IgM+IgD+CD27+ B Cells and Their Frequencies in Appendectomized and/or Tonsillectomized Individuals.

BACKGROUND: In humans, blood circulating IgM+IgD+CD27+ B cells are considered analogous to those described in the marginal zone of the spleen and are involved in important immunological processes. The homing receptors they express, and the organs involved in their development (for example, intestinal organs in rabbits) are only partially known. We recently reported that this population is heterogeneous and composed of at least two subsets: one expressing high levels of IgM - IgMhi B cells - and another low levels - IgMlo B cells. OBJECTIVES: To evaluate the expression of homing receptors on IgD+CD27+ IgMhi and IgMlo B cells and quantify their frequencies in blood of control and appendectomized and/or tonsillectomized volunteers. MATERIALS AND METHODS: Using spectral flow cytometry, the simultaneous expression of 12 previously reported markers that differentiate IgMhi B cells and IgMlo B cells and of α4ß7, CCR9, CD22 and CCR10 were evaluated in blood circulating B cells of control and appendectomized and/or tonsillectomized volunteers. RESULTS: The existence of phenotypically defined IgMlo and IgMhi B cell subsets was confirmed. They differentially expressed intestinal homing receptors, and the expression of α4ß7 and CCR9 seems to determine new IgM subpopulations. IgMlo and IgMhi B cells were detected at lower frequencies in the appendectomized and/or tonsillectomized volunteers relative to controls. CONCLUSIONS: Human blood circulating IgD+CD27+ IgMlo and IgMhi B cell subsets differentially express homing receptors, and it is necessary to investigate if mucosal organs are important in their development.

Seroprevalence and seroconversion rates to SARS-CoV-2 in interns, residents, and medical doctors in a University Hospital in Bogotá, Colombia / Tasas de seroprevalencia y seroconversión al SARS-CoV-2 en internos, residentes y médicos en un Hospital Universitario de Bogotá, Colombia

Abstract Objectives: To determine the prevalence of antibodies to SARS-CoV-2 and the incidence of seroconversion in the first month of follow-up among interns, residents, and medical doctors attending patients at a University Hospital in Bogota (Colombia). Design or methods: A cross-sectional and a prospective study were performed during June, July, and August 2020 to assess seroprevalence and seroconversion rates using CLIA IgG for SARS-CoV-2. LFA IgG and IgM and ELFA IgM were also determined to explore concordance with CLIA IgG. Results: At baseline, 8 (2.28% 95%CI 1.16-4.43%) participants were IgG positive for SARS-CoV-2 by CLIA. At the end of the study, 21 (5.98% 95%CI 3.94-8.97%) individuals seroconverted by CLIA IgG. In all, 29 individuals had IgG by CLIA and of these 11 (3.13% 95%CI 1.76-5.52%) were asymptomatic. No associations with risk factors for infection were identified. CLIA IgG had moderate concordance (>962 samples) with LFA IgG and ELFA IgM, but minimal with LFA IgM. Conclusions: Our report is the first in Latina America on seroprevalence and seroconversion rates in medical healthcare workers. The relatively high rate (>3%) of asymptomatic health care workers with evidence of previous SARS-CoV-2 infection underscores the need to screen this population for infection to prevent infection/disease spread.

Resumen Objetivos: Determinar la prevalencia de anticuerpos frente al SARS-CoV-2 y la incidencia de seroconversión en el primer mes de seguimiento en internos, residentes y médicos que atienden pacientes en un Hospital Universitario de Bogotá (Colombia). Diseño y métodos: Se realizó un estudio transversal y prospectivo durante junio, julio y agosto de 2020 para evaluar las tasas de seroprevalencia y seroconversión utilizando CLIA IgG para SARS-CoV-2. También se determinaron LFA IgG e IgM y ELFA IgM para explorar la concordancia con CLIA IgG. Resultados: Al inicio del estudio, 8 (2,28% IC del 95% 1,16-4,43%) participantes fueron IgG positivos para SARS-CoV-2 por CLIA. Al final del estudio, 21 (5,98% IC 95% 3,94-8,97%) individuos seroconvirtieron por CLIA IgG. En total, 29 individuos tenían IgG por CLIA y de estos 11 (3,13% 95% IC 1,76-5,52%) eran asintomáticos. No se identificaron asociaciones con factores de riesgo de infección. El CLIA IgG tuvo una concordancia moderada (> 962 muestras) con LFA IgG y ELFA IgM, pero mínima con el LFA IgM. Conclusiones: Nuestro informe es el primero en América Latina sobre tasas de seroprevalencia y seroconversión en trabajadores médicos de la salud. La tasa relativamente alta (> 3%) de trabajadores de la salud asintomáticos con evidencia de infección previa por SARS-CoV-2 resalta la necesidad de realizar pruebas de detección de infección en esta población para prevenir la propagación de la infección.

Differential Expression of IgM and IgD Discriminates Two Subpopulations of Human Circulating IgM+IgD+CD27+ B Cells That Differ Phenotypically, Functionally, and Genetically.

The origin and function of blood IgM+IgD+CD27+ B cells is controversial, and they are considered a heterogeneous population. Previous staining of circulating B cells of healthy donors with rotavirus fluorescent virus-like particles allowed us to differentiate two subsets of IgM+IgD+CD27+: IgMhi and IgMlo B cells. Here, we confirmed this finding and compared the phenotype, transcriptome, in vitro function, and Ig gene repertoire of these two subsets. Eleven markers phenotypically discriminated both subsets (CD1c, CD69, IL21R, CD27, MTG, CD45RB, CD5, CD184, CD23, BAFFR, and CD38) with the IgMhi phenotypically resembling previously reported marginal zone B cells and the IgMlo resembling both naïve and memory B cells. Transcriptomic analysis showed that both subpopulations clustered close to germinal center-experienced IgM only B cells with a Principal Component Analysis, but differed in expression of 78 genes. Moreover, IgMhi B cells expressed genes characteristic of previously reported marginal zone B cells. After stimulation with CpG and cytokines, significantly (p < 0.05) higher frequencies (62.5%) of IgMhi B cells proliferated, compared with IgMlo B cells (35.37%), and differentiated to antibody secreting cells (14.22% for IgMhi and 7.19% for IgMlo). IgMhi B cells had significantly (p < 0.0007) higher frequencies of mutations in IGHV and IGKV regions, IgMlo B cells had higher usage of IGHJ6 genes (p < 0.0001), and both subsets differed in their HCDR3 properties. IgMhi B cells shared most of their shared IGH clonotypes with IgM only memory B cells, and IgMlo B cells with IgMhi B cells. These results support the notion that differential expression of IgM and IgD discriminates two subpopulations of human circulating IgM+IgD+CD27+ B cells, with the IgMhi B cells having similarities with previously described marginal zone B cells that passed through germinal centers, and the IgMlo B cells being the least differentiated amongst the IgM+CD27+ subsets.

Procedimientos de mama guiados por resonancia magnética: nuestra experiencia / MRI Breast Guided Procedures: Our Experience

La resonancia magnética (RM) de seno es la modalidad diagnóstica más sensible para la detección de cáncer de seno; sin embargo, su especificidad es limitada, pues varía entre el 40 y 80 %. Esto se debe a las características de realce de algunas lesiones benignas que se sobreponen a las de lesiones malignas, y para cuyo diagnóstico se requiere análisis histológico. Para el diagnóstico histológico de estas lesiones ­que solo se visualizan por RM y no por otro método diagnóstico­ los procedimientos guiados por RM son la elección. La biopsia guiada por RM se utiliza en pacientes con ultrasonido dirigido negativo. El ROLL (Radioguided Occult Lesión Localization) es una técnica alternativa a la marcación prequirúrgica con alambre de lesiones ocultas y ha sido más ampliamente utilizada en ultrasonido que en RM, con muy buenos resultados. Se obtuvieron de manera retrospectiva los datos de las pacientes que ingresaron al sistema de nuestra institución para la realización de biopsia de seno y marcación de seno guiada por RM. Posteriormente se recopilaron las patologías. Los resultados se tabularon en Excel para su análisis.

Breast magnetic resonance imaging (MRI) is the most sensitive diagnostic modality for the detection of breast cancer; however, its specificity is limited, ranging from 40% to 80%. This is due to the enhancement characteristics of some benign lesions that overlap those of malignant lesions, for which histology is required for diagnosis. For the histological diagnosis of these lesions, which are only visualized by MRI and not by any other diagnostic method, MR-guided procedures are the choice. MR-guided biopsy is used in patients with negative directed ultrasound. ROLL (Radioguided Occult Injury Localization) is an alternative technique to preoperative wire marking of hidden lesions and has been more widely used in ultrasound than in MRI, with very good results. Retrospectively the data of all patients admitted to our center for Breast MRI biopsy and Breast MRI localizations were retrieved. Pathologies were then collected and the results were tabulated in Excel for analysis.

Identification and Characterization at the Single-Cell Level of Cytokine-Producing Circulating Cells in Children With Dengue.

In this study, we identified, at the single-cell level, naturally induced cytokine-producing circulating cells (CPCCs) in children with dengue virus (DENV) infection ranging clinically from mild to severe disease. Tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) CPCCs were detected in children with primary or secondary acute dengue virus (DENV) infection, and the pattern of these cytokines was similar to that seen in the supernatant of cultured peripheral blood mononuclear cells and partially comparable to that found in plasma. Monocytes, B cells, and myeloid dendritic cells (mDCs) were the primary CPCCs detected, and the frequency of mDCs was significantly higher in severe disease. B cells isolated from children with dengue spontaneously secreted TNF-α, IL-6, and interleukin 10, and supernatants from cultures of purified B cells induced activation of allogeneic T cells, supporting an antibody-independent function of these cells during DENV infection. Thus, CPCCs could be a new immune parameter with potential use to evaluate pathogenesis in this infection.

Total and Envelope Protein-Specific Antibody-Secreting Cell Response in Pediatric Dengue Is Highly Modulated by Age and Subsequent Infections.

The response of antibody-secreting cells (ASC) induced by dengue has only recently started to be characterized. We propose that young age and previous infections could be simple factors that affect this response. Here, we evaluated the primary and secondary responses of circulating ASC in infants (6-12 months old) and children (1-14 years old) infected with dengue showing different degrees of clinical severity. The ASC response was delayed and of lower magnitude in infants, compared with older children. In primary infection (PI), the total and envelope (E) protein-specific IgM ASC were dominant in infants but not in children, and a negative correlation was found between age and the number of IgM ASC (rho = -0.59, P = 0.03). However, infants with plasma dengue-specific IgG detectable in the acute phase developed an intense ASC response largely dominated by IgG and comparable to that of children with secondary infection (SI). IgM and IgG produced by ASC circulating in PI or SI were highly cross-reactive among the four serotypes. Dengue infection caused the disturbance of B cell subsets, particularly a decrease in the relative frequency of naïve B cells. Higher frequencies of total and E protein-specific IgM ASC in the infants and IgG in the children were associated with clinically severe forms of infection. Therefore, the ASC response induced by dengue is highly influenced by the age at which infection occurs and previous immune status, and its magnitude is a relevant element in the clinical outcome. These results are important in the search for correlates of protection and for determining the ideal age for vaccinating against dengue.

Rapid Proliferation and Differentiation of a Subset of Circulating IgM Memory B Cells to a CpG/Cytokine Stimulus In Vitro.

Circulating human IgM expressing memory B cells have been incompletely characterized. Here, we compared the phenotype and in vitro functional response (capacity to proliferate and differentiate to antibody secreting cells) in response to CpG and a cytokine cocktail (IL-2, IL-6, and IL-10) of sorted naïve B cells, IgM memory B cells and isotype-switched circulating memory B cells. Compared to naïve B cells, IgM memory B cells had lower integrated mean fluorescence intensity (iMFI) of BAFF-R, CD38, CD73, and IL-21R, but higher iMFI of CD95, CD11c, TLR9, PD-1, and CD122. Compared to switched memory B cells, IgM memory B cells had higher iMFI of BAFF-R, PD-1, IL-21R, TLR9, and CD122, but lower iMFI of CD38, CD95, and CD73. Four days after receiving the CpG/cytokine cocktail, higher frequencies of IgM than switched memory B cells-and these in turn greater than naïve cells-proliferated and differentiated to antibody secreting cells. At this time point, a small percentage (median of 7.6%) of stimulated IgM memory B cells changed isotype to IgG. Thus, among the heterogeneous population of human circulating IgM memory B cells a subset is capable of a rapid functional response to a CpG/cytokine stimulus in vitro.

Caco-2 cells infected with rotavirus release extracellular vesicles that express markers of apoptotic bodies and exosomes.

Previously, we showed that infecting human intestinal epithelial cells (Caco-2) with rotavirus (RV) increases the release of extracellular vesicles (EVs) with an immunomodulatory function that, upon concentration at 100,000×g, present buoyant densities on a sucrose gradient of between 1.10 to 1.18 g/ml (characteristic of exosomes) and higher than 1.24 g/ml (proposed for apoptotic bodies). The effect of cellular death induced by RV on the composition of these EV is unknown. Here, we evaluated exosome (CD63, Hsc70, and AChE) and apoptotic body (histone H3) markers in EVs isolated by differential centrifugation (4000×g, 10,000×g, and 100,000×g) or filtration/ultracentrifugation (100,000×g) protocols. When we infected cells in the presence of caspase inhibitors, Hsc70 and AChE diminished in EVs obtained at 100,000×g, but not in EVs obtained at 4000×g or 10,000×g. In addition, caspase inhibitors decreased CD63 and AChE in vesicles with low and high buoyant densities. Without caspase inhibitors, RV infection increased exosome markers in all of the EVs obtained by differential centrifugation. However, CD63 preferentially localized in the 100,000×g fraction and H3 only increased in EVs concentrated at 100,000×g and with high buoyant densities on a sucrose gradient. Thus, RV infection increases the release of EVs that, upon concentration at 100,000×g, are composed by exosomes and apoptotic bodies, which can partially be separated using sucrose gradients.

Simultaneous assessment of rotavirus-specific memory B cells and serological memory after B cell depletion therapy with rituximab.

The mechanisms that contribute to the maintenance of serological memory are still unclear. Rotavirus (RV) memory B cells (mBc) are enriched in IgM(+) and CD27- subpopulations, which are associated with autoimmune diseases pathogenesis. In patients with autoimmune diseases treated with Rituximab (RTX), some autoantibodies (auto-Abs) decrease after treatment, but other auto-Abs and pathogen-specific IgG Abs remain unchanged. Thus, maintenance of autoimmune and pathogen-specific serological memory may depend on the type of antigen and/or Ab isotype evaluated. Antigen-specific mBc and antigen-specific Abs of different isotypes have not been simultaneously assessed in patients after RTX treatment. To study the relationship between mBc subpopulations and serological memory we characterized total, RV- and tetanus toxoid (TT)-specific mBc by flow cytometry in patients with autoimmune diseases before and after treatment with RTX. We also measured total, RV- and TT-Abs, and some auto-Abs by kinetic nephelometry, ELISA, and EliA tests, respectively. Minor differences were observed between the relative frequencies of RV-mBc in healthy controls and patients with autoimmune disease. After RTX treatment, naïve Bc and total, RV- and TT-specific mBc [IgM(+), switched (IgA(+)/IgG(+)), IgM(+) only, IgD(+) only, and CD27- (IgA(+)/IgG(+)/IgM(+))] were significantly diminished. An important decrease in total plasma IgM and minor decreases in total IgG and IgA levels were also observed. IgM rheumatoid factor, IgG anti-CCP, and IgG anti-dsDNA were significantly diminished. In contrast, RV-IgA, RV-IgG and RV-IgG1, and TT-IgG titers remained stable. In conclusion, in patients with autoimmunity, serological memory against RV and TT seem to be maintained by long-lived plasma cells, unaffected by RTX, and an important proportion of total IgM and serological memory against some auto-antigens seem to be maintained by short-lived plasma cells, dependent on mBc precursors depleted by RTX.

Human myeloid dendritic cells treated with supernatants of rotavirus infected Caco-2 cells induce a poor Th1 response.

We have previously shown that human myeloid dendritic cells treated with purified rotavirus induce an allogenic Th1 response. To determine if rotavirus in the context of an intestinal microenvironment modulates the function of dendritic cells, we treated these cells with supernatants from non-infected or infected Caco-2 cells and studied their capacity to promote Th1 or Th2 responses. Dendritic cells treated with supernatants from rotavirus-infected Caco-2 cells promoted a significantly lower Th1 response, in comparison with those treated with purified rotavirus. We wanted to establish if TGF-ß1, induced, or TSLP, not induced, during rotavirus infection, could mediate this effect. Neutralization of TGF-ß but not TSLP in the supernatant prior to treatment of dendritic cells increased their capacity to promote a Th1 response. The results suggest that the TGF-ß1 induced by rotavirus could be an immune evasion mechanism, and may partially explain the poor rotavirus-specific T cell response we have previously evidenced.

Membrane vesicles released by intestinal epithelial cells infected with rotavirus inhibit T-cell function.

Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and "danger signals" released by these cells may modulate viral immunity. We have recently shown that human model IEC (Caco-2 cells) infected with rhesus-RV release a non-inflammatory group of immunomodulators that includes heat shock proteins (HSPs) and TGF-ß1. Here we show that both proteins are released in part in association with membrane vesicles (MV) obtained from filtrated Caco-2 supernatants concentrated by ultracentrifugation. These MV express markers of exosomes (CD63 and others), but not of the endoplasmic reticulum (ER) or nuclei. Larger quantities of proteins associated with MV were released by RV-infected cells than by non-infected cells. VP6 co-immunoprecipitated with CD63 present in these MV, and VP6 co-localized with CD63 in RV-infected cells, suggesting that this viral protein is associated with the MV, and that this association occurs intracellularly. CD63 present in MV preparations from stool samples from 36 children with gastroenteritis due or not due to RV were analyzed. VP6 co-immunoprecipitated with CD63 in 3/8 stool samples from RV-infected children, suggesting that these MV are released by RV-infected cells in vivo. Moreover, fractions that contained MV from RV-infected cells induced death and inhibited proliferation of CD4(+) T cells to a greater extent than fractions from non-infected cells. These effects were in part due to TGF-ß, because they were reversed by treatment of the T cells with the TGF-ß-receptor inhibitor ALK5i. MV from RV-infected and non-infected cells were heterogeneous, with morphologies and typical flotation densities described for exosomes (between 1.10 and 1.18 g/mL), and denser vesicles (>1.24 g/mL). Both types of MV from RV-infected cells were more efficient at inhibiting T-cell function than were those from non-infected cells. We propose that RV infection of IEC releases MV that modulate viral immunity.

Rotavirus differentially infects and polyclonally stimulates human B cells depending on their differentiation state and tissue of origin.

We have shown previously that rotavirus (RV) can infect murine intestinal B220(+) cells in vivo (M. Fenaux, M. A. Cuadras, N. Feng, M. Jaimes, and H. B. Greenberg, J. Virol. 80:5219-5232, 2006) and human blood B cells in vitro (M. C. Mesa, L. S. Rodriguez, M. A. Franco, and J. Angel, Virology 366:174-184, 2007). However, the effect of RV on B cells, especially those present in the human intestine, the primary site of RV infection, is unknown. Here, we compared the effects of the in vitro RV infection of human circulating (CBC) and intestinal B cells (IBC). RV infected four times more IBC than CBC, and in both types of B cells the viral replication was highly restricted to the memory subset. RV induced cell death in 30 and 3% of infected CBC and IBC, respectively. Moreover, RV induced activation and differentiation into antibody-secreting cells (ASC) of CBC but not IBC when the B cells were present with other mononuclear cells. However, RV did not induce these effects in purified CBC or IBC, suggesting the participation of other cells in activating and differentiating CBC. RV infection was associated with enhanced interleukin-6 (IL-6) production by CBC independent of viral replication. The infection of the anti-B-cell receptor, lipopolysaccharide, or CpG-stimulated CBC reduced the secretion of IL-6 and IL-8 and decreased the number of ASC. These inhibitory effects were associated with an increase in viral replication and cell death and were observed in polyclonally stimulated CBC but not in IBC. Thus, RV differentially interacts with primary human B cells depending on their tissue of origin and differentiation stage, and it affects their capacity to modulate the local and systemic immune responses.

Characterization of rotavirus specific B cells and their relation with serological memory.

We quantified circulating total, rotavirus (RV) and Tetanus toxin (TT) memory B cells (mBc) in healthy adults using a limiting dilution assay (LDA) and a flow cytometry assay (FCA) that permit evaluation of both CD27+ and CD27- mBc. RV mBc were enriched in the CD27-, IgG+ and in the CD27+, IgM+ subsets. The numbers of RV mBc were higher by FCA than by LDA and results of the two assays did not correlate. TT IgGmBc and RV IgA mBc determined by FCA and by LDA correlated with TT plasma IgG and RV plasma IgA, respectively. The mean ratio of specific mBc/mug/ml of the corresponding plasma immunoglobulin was lower for TT IgG than for RV IgA mBc. Our studies contribute to understand the relationship between circulating mBc and serological memory, and enhance our capacity to develop better correlates of protection against RV disease.

Evaluation of circulating intestinally committed memory B cells in children vaccinated with attenuated human rotavirus vaccine.

In a double blind trial, 319 fully immunized children received two doses of either placebo or 10(6.7) focus-forming units of the attenuated RIX4414 human rotavirus (RV) vaccine ("all-in-one" formulation). Plasma RV-specific IgA (RV IgA), stool RV IgA, and circulating total and RV memory B cells (CD19+ IgD+/- CD27+) with an intestinal homing phenotype (alpha4beta7+ CCR9+/-) were measured, after the first and second doses, as potential correlates of protection. After the first and/or second dose, 54% of vaccinees and 13% of placebo recipients had plasma RV IgA. Before vaccination, most (95%) of the children (of both study groups) were breast-fed and had stool RV IgA (68.64%). Coproconversion (4-fold increase) after the first and/or second dose was observed in 32.7% of vaccinees and 17.4% of placebo recipients. No significant difference was seen when comparing the frequencies of any subset of memory B cells between vaccinees and placebo recipients. Statistically significant weak correlations were found between plasma RV IgA titers and coproconversion, and several subsets of memory B cells. The vaccine provided 74.8% protection (95% confidence interval, 30.93-92.62) against any RV gastroenteritis and 100% protection (95% confidence interval, 14.53-100) against severe RV gastroenteritis. When vaccinees and placebo recipients were considered together, a correlation was found between protection from disease and plasma RV IgA measured after dose 2 and RV memory (IgD- CD27+ alpha4beta7+ CCR9+) circulating B cells measured after dose 1. However, the correlation coefficients for both tests were low (<0.2), suggesting that other factors are important in explaining protection from disease.

Interaction of rotavirus with human peripheral blood mononuclear cells: plasmacytoid dendritic cells play a role in stimulating memory rotavirus specific T cells in vitro.

We studied the interaction of RV with human peripheral blood mononuclear cells (PBMC) from adult volunteers. After exposure of PBMC to rhesus RV (RRV), T and B lymphocytes, NK cells, monocytes, and myeloid and plasmacytoid dendritic cells expressed RV non-structural proteins, at variable levels. Expression of these RV proteins was abolished if infection was done in the presence of anti-VP7 neutralizing antibodies or 10% autologous serum. Supernatants of RRV exposed PBMC contained TNF-alpha, IL-6, IFN-alpha, IFN-gamma, IL-2 and IL-10. Plasmacytoid DC were found to be the main source of IFN-alpha production, and in their absence the production of IFN-gamma and the frequency of RV specific T cells that secrete IFN-gamma diminished. Finally, we could not detect RV-antigen associated with the PBMC or expression of RV non-structural proteins in PBMC of acutely RV-infected children. Thus, although PBMC are susceptible to the initial steps of RV infection, most PBMC of children with RV-gastroenteritis are not infected.

Immunity and correlates of protection for rotavirus vaccines.

Rotaviruses are the most common cause of severe, dehydrating diarrhea in children worldwide. The tremendous global incidence of rotavirus gastroenteritis, especially in developing countries, emphasizes the need for vaccines to prevent associated morbidity and mortality. However, immunity to rotavirus is not completely understood. At this time, total serum RV IgA, measured shortly after infection, appears to be the best marker of protection against rotavirus. This review describes the current understanding of rotavirus immunity, including mechanisms of protection against rotavirus from selected animal models, and correlates of protection associated with natural infection or vaccination from humans.

Interaction of rotavirus with human myeloid dendritic cells.

We have previously shown that very few rotavirus (RV)-specific T cells that secrete gamma interferon circulate in recently infected and seropositive adults and children. Here, we have studied the interaction of RV with myeloid immature (IDC) and mature dendritic cells (MDC) in vitro. RV did not induce cell death of IDC or MDC and induced maturation of between 12 and 48% of IDC. Nonetheless, RV did not inhibit the maturation of IDC or change the expression of maturation markers on MDC. After treatment with RV, few IDC expressed the nonstructural viral protein NSP4. In contrast, a discrete productive viral infection was shown in MDC of a subset of volunteers, and between 3 and 46% of these cells expressed NSP4. RV-treated IDC secreted interleukin 6 (IL-6) (but not IL-1beta, IL-8, IL-10, IL-12, tumor necrosis factor alpha, or transforming growth factor beta), and MDC released IL-6 and small amounts of IL-10 and IL-12p70. The patterns of cytokines secreted by T cells stimulated by staphylococcal enterotoxin B presented by MDC infected with RV or uninfected were comparable. The frequencies and patterns of cytokines secreted by memory RV-specific T cells evidenced after stimulation of peripheral blood mononuclear cells (PBMC) with RV were similar to those evidenced after stimulation of PBMC with RV-infected MDC. Finally, IDC treated with RV strongly stimulated naive allogeneic CD4+ T cells to secrete Th1 cytokines. Thus, although RV does not seem to be a strong maturing stimulus for DC, it promotes their capacity to prime Th1 cells.