Signaling via the MyD88 adaptor protein in B cells suppresses protective immunity during Salmonella typhimurium infection.

The myeloid differentiation primary response gene 88 (Myd88) is critical for protection against pathogens. However, we demonstrate here that MyD88 expression in B cells inhibits resistance of mice to Salmonella typhimurium infection. Selective deficiency of Myd88 in B cells improved control of bacterial replication and prolonged survival of the infected mice. The B cell-mediated suppressive pathway was even more striking after secondary challenge. Upon vaccination, mice lacking Myd88 in B cells became completely resistant against this otherwise lethal infection, whereas control mice were only partially protected. Analysis of immune defenses revealed that MyD88 signaling in B cells suppressed three crucial arms of protective immunity: neutrophils, natural killer cells, and inflammatory T cells. We further show that interleukin-10 is an essential mediator of these inhibitory functions of B cells. Collectively, our data identify a role for MyD88 and B cells in regulation of cellular mechanisms of protective immunity during infection.

Impact of retroviral vector components stoichiometry on packaging cell lines: effects on productivity and vector quality.

The productivity of retroviral vector producer cell lines is currently low; this can be attributed partly to disruption of the viral components stoichiometry originated by the dissociation of gag-pol, env, and viral genome. This paper addresses the impact of viral components stoichiometry on cell productivity and vector quality (vector stability and transduction efficiency). A strategy is proposed for the development of packaging cell lines allowing for stoichiometric optimization of viral components. This strategy is based on the introduction of a transgene in the first step of cell line development, thus making it possible to eliminate transgene limitations and therefore exploring maximal infectious productivity based on the levels of expression of Gag-Pol and envelope. The transgene can subsequently be exchanged for a gene of interest, using site-specific flipase/flipase recombination target (Flp/FRT) cassette replacement. It was observed that Gag-Pol controlled the order of magnitude of infectious titer: a small, 2-fold variation in its expression could result in a 10- to 100-fold improvement in infectious titer; the RNA transgene and envelope expression limitations had a lower impact. The ratios of retroviral components strongly affected cell productivity but did not affect vector stability; however, transduction efficiencies were affected by an imbalanced ratio of the components, resulting in the production of higher amounts of defective virus. Thus, stoichiometric optimization of viral components not only improves cell productivity but also increases vector quality.

Stochastic monoallelic expression of IL-10 in T cells.

IL-10 is a potent anti-inflammatory and immunomodulatory cytokine, exerting major effects in the degree and quality of the immune response. Using a newly generated IL-10 reporter mouse model, which easily allows the study of IL-10 expression from each allele in a single cell, we report here for the first time that IL-10 is predominantly monoallelic expressed in CD4+ T cells. Furthermore, we have compelling evidence that this expression pattern is not due to parental imprinting, allelic exclusion, or strong allelic bias. Instead, our results support a stochastic regulation mechanism, in which the probability to initiate allelic transcription depends on the strength of TCR signaling and subsequent capacity to overcome restrictions imposed by chromatin hypoacetylation. In vivo Ag-experienced T cells show a higher basal probability to transcribe IL-10 when compared with naive cells, yet still show mostly monoallelic IL-10 expression. Finally, statistical analysis on allelic expression data shows transcriptional independence between both alleles. We conclude that CD4+ T cells have a low probability for IL-10 allelic activation resulting in a predominantly monoallelic expression pattern, and that IL-10 expression appears to be stochastically regulated by controlling the frequency of expressing cells, rather than absolute protein levels per cell.

Transcriptional profiling of Arabidopsis tissues reveals the unique characteristics of the pollen transcriptome.

Pollen tubes are a good model for the study of cell growth and morphogenesis because of their extreme elongation without cell division. Yet, knowledge about the genetic basis of pollen germination and tube growth is still lagging behind advances in pollen physiology and biochemistry. In an effort to reduce this gap, we have developed a new method to obtain highly purified, hydrated pollen grains of Arabidopsis through flowcytometric sorting, and we used GeneChips (Affymetrix, Santa Clara, CA; representing approximately 8,200 genes) to compare the transcriptional profile of sorted pollen with those of four vegetative tissues (seedlings, leaves, roots, and siliques). We present a new graphical tool allowing genomic scale visualization of the unique transcriptional profile of pollen. The 1,584 genes expressed in pollen showed a 90% overlap with genes expressed in these vegetative tissues, whereas one-third of the genes constitutively expressed in the vegetative tissues were not expressed in pollen. Among the 469 genes enriched in pollen, 162 were selectively expressed, and most of these had not been associated previously with pollen. Their functional classification reveals several new candidate genes, mainly in the categories of signal transduction and cell wall biosynthesis and regulation. Thus, the results presented improve our knowledge of the molecular mechanisms underlying pollen germination and tube growth and provide new directions for deciphering their genetic basis. Because pollen expresses about one-third of the number of genes expressed on average in other organs, it may constitute an ideal system to study fundamental mechanisms of cell biology and, by omission, of cell division.

Selective gene expression of latent murine gammaherpesvirus 68 in B lymphocytes.

Intranasal infection of mice with murine gammaherpesvirus 68 (MHV-68), a virus genetically related to the human pathogen Kaposi's sarcoma-associated herpesvirus, results in a persistent, latent infection in the spleen and other lymphoid organs. Here, we have determined the frequency of virus infection in splenic dendritic cells, macrophages, and several B-cell subpopulations, and we quantified cell type-dependent virus transcription patterns. The frequencies of virus genome positive cells were maximal at 14 days postinfection in all splenic cell populations analyzed. Marginal zone and germinal center B cells harbored the highest frequency of infection and the former population accounted for approximately half the total number of infected B cells. Analysis of virus transcription during the establishment of latency revealed that virus gene expression in B cells was restricted and dependent on the differentiation stage of the B cell. Notably, transcription of ORF73 was detected in germinal center B cells, a finding in agreement with the predicted latent genome maintenance function of ORF73 in dividing cells. At late times after infection, virus DNA could only be detected in newly formed and germinal center B cells, which suggests that B cells play a critical role in facilitating life-long latency.

Cell growth arrest by nucleotides, nucleosides and bases as a tool for improved production of recombinant proteins.

Arresting cell growth and thus decreasing cell division potentially lessens the chance for genetic drift in the cell population; this would be of utmost importance for the consistent production of biopharmaceuticals during long periods. The drawback of the addition of well-known synchronizing agents, such as chemotherapeutics, is that they cause a disproportionate accumulation of cellular constituents, leading to cell death. The use of compounds that are naturally synthesized by the cell, as is the case of nucleotides, nucleosides, and bases (Nt/Ns/B), is shown in this work to be a promising tool. The addition of purines and pyrimidines was tested using a CHO cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP). From the chemical alternatives tested, AMP was the most promising compound for protein production improvement; it reduced cell growth and maintained the culture with high cell viability for long periods, while increasing SEAP specific productivity 3-fold. The use of CHO and BHK mammalian cells producing Factor VII and the use of a insect cell line (Sf9) showed that the effect of AMP addition seems to be independent of the r-protein and cell line. With the addition of AMP, accumulation of cells at the S phase was accompanied by an increase of the protein specific productivity. Addition of known synchronizing drugs (aphidicolin and doxorubicin) and application of environmental cell growth arrest strategies (depletion of nutrients and byproduct accumulation) showed also to effectively arrest CHO cell growth. A careful look onto cell cycle distribution in the different scenarios created, shows whether it is important to consider r-protein expression dependency upon cell cycle in process optimization and operation strategies.

Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide.

Regulatory CD4 T cells (Treg) control inflammatory reactions to commensal bacteria and opportunist pathogens. Activation of Treg functions during these processes might be mediated by host-derived proinflammatory molecules or directly by bacterial products. We tested the hypothesis that engagement of germline-encoded receptors expressed by Treg participate in the triggering of their function. We report that the subset of CD4 cells known to exert regulatory functions in vivo (CD45RB(low) CD25(+)) selectively express Toll-like receptors (TLR)-4, -5, -7, and -8. Exposure of CD4(+) CD25(+) cells to the TLR-4 ligand lipopolysaccharide (LPS) induces up-regulation of several activation markers and enhances their survival/proliferation. This proliferative response does not require antigen-presenting cells and is augmented by T cell receptor triggering and interleukin 2 stimulation. Most importantly, LPS treatment increases CD4(+) CD25(+) cell suppressor efficiency by 10-fold and reveals suppressive activity in the CD4(+) CD45RB(low) CD25(-) subset that when tested ex-vivo, scores negative. Moreover, LPS-activated Treg efficiently control naive CD4 T cell-dependent wasting disease. These findings provide the first evidence that Treg respond directly to proinflammatory bacterial products, a mechanism that likely contributes to the control of inflammatory responses.

Specificity requirements for selection and effector functions of CD25+4+ regulatory T cells in anti-myelin basic protein T cell receptor transgenic mice.

CD25(+)4(+) regulatory T cells (T(reg)) play an indispensable role in preventing autoimmunity. Little is known, however, about the antigen specificities required for their development and effector functions. Mice transgenic for an anti-myelin basic protein (MBP) T cell antigen receptor (TCR) spontaneously develop experimental autoimmune encephalomyelitis (EAE) when deficient for the RAG-1 gene (T/R(-)), whereas RAG-1-competent transgenic animals (T/R(+)) remain healthy, protected by CD4(+) T(reg)-expressing endogenous TCRs. We have now investigated the role and specificity of CD25(+)4(+) T(reg) in this system. The results show that T/R(+) animals contain MBP-specific suppressive CD25(+)4(+) cells, whereas T/R(-) do not. Adoptive transfer of CD25(+)4(+) cells from nontransgenic or T/R(+) donors into T/R(-) mice prevented the development of EAE. Surprisingly, transfer of nontransgenic CD25(+)4(+) cells purified from T/R(+) donors conferred only a limited protection, possibly because of their restricted repertoire diversity that we demonstrate here. Absence of transgenic CD25(+)4(+) cells in animals deficient for endogenous TCRalpha chains and analyses of endogenous TCR gene expression in subsets of CD4(+) cells from T/R(+) mice demonstrate that development of transgenic MBP-specific CD25(+)4(+) T(reg) depends on the coexpression of endogenous TCRalpha chains. Taken together, these results indicate that specificity to MBP is required for effector functions but is not sufficient for thymic selection/commitment of CD25(+)4(+) T(reg) preventing EAE.

Peripheral expansion of thymus-derived regulatory cells in anti-myelin basic protein T cell receptor transgenic mice.

CD4+ regulatory T cells (Treg) play an indispensable role in tolerance to peripheral antigens, but the origin of the Treg pool in the adult remains unclear. Thus, while thymic commitment of Treg has been demonstrated, evidence also exists for the peripheral recruitment of naïve tissue-specific T cells into Treg functions. Anti-myelin basic protein TCR transgenic mice spontaneously develop autoimmune encephalomyelitis when "monoclonal", but are protected by adoptive transfer of CD4+ cells from wild-type donors. We have now used this transfer system to investigate whether previously infused Treg can recruit transgenic T cells to regulatory functions. The results show that transgenic T cells from protected animals did not transfer tolerance to secondary recipients, and that elimination of donor Treg in protected recipients resulted in rapid onset of disease. In addition, Treg-containing T cell susbsets were highly enriched for proliferating cells in vivo, which was also the case for CD4+CD25+ T cells in normal animals. These observations thus exclude peripheral differentiation of Treg in this particular system, and indicate that expansion of thymically committed cells ensures the maintenance of the peripheral Treg pool in the adult.