Use of Lactobacillus to prevent infection by pathogenic bacteria

This review focuses on the use and potential of Lactobacillus to prevent infections of the urogenital and intestinal tracts. The presence and dominance of Lactobacillus in the vagina is associated with a reduced risk of bacterial vaginosis and urinary tract infections. The mechanisms appear to involve anti-adhesion factors, by-products such as hydrogen peroxide and bacteriocins lethal to pathogens, and perhaps immune modulation or signaling effects. The instillation of Lactobacillus GR-1 and B-54 or RC-14 strains into the vagina has been shown to reduce the risk of urinary tract infections, and improve the maintenance of a normal flora. Ingestion of these strains into the gut has also been shown to modify the vaginal flora to a more healthy state. In addition, these strains inhibit the growth of intestinal, as well as urogenital pathogens, colonize the gut and protect against infections as shown in mice. Other probiotic strains, such as Lactobacillus GG, have been shown to prevent and treat gastroenteritis caused by rotavirus and bacteria. Given that lactobacilli are not the dominant commensals in a gut which comprises around 10(10) organisms, much work is still needed to define the mechanisms whereby GR-1, RC-14, GG and other strains contribute to health restoration and maintenance. Such critically important studies will require the medical science community to show a willingness to turn away from pharmaceutical remedies as the only solution to health and disease.     

Cytolytic T cells activated by H-2-controlled E molecules cross-react with A molecules

Cell-mediated lymphocytotoxicity was generated in four strain combinations differing only by the cell-surface expression of the class II E molecule controlled by the H-2 complex. The four combinations were: B10.D2(R107) anti-B10.A(3R), B10.A(4R) anti-B10.A(2R), B10.GD anti-B10.D2(R101), and B10.S(7R) anti-B10.S(9R). In all four of these combinations, the stimulator expresses E molecules on the cell surface, while the responder does not. The cytolytic T lymphocytes generated in the B10.D2(R107) anti-B10.A(3R) and B10.A(4R) anti-B10.A(2R) combinations reacted not only with the stimulator but also with strains that do not express cell-surface E molecules, in particular, strains carrying the H-2 ^f and H-2 ^q haplotypes. The cross-reactivity with E-negative strains could be blocked by monoclonal antibodies specific for the A^f or A^q molecules but not by antibodies recognizing determinants on E or class I (K) molecules. The anti-H-2^f cross-reactivity could be inhibited by H-2 ^q cold targets and, reciprocally, the anti-H-2^q reactivity could be blocked by H-2 ^f cold targets. These findings are interpreted as indicating that the cytolytic T lymphocytes stimulated by E molecules can recognize and lyse cells lacking E molecules but expressing A molecules. The observed E-A cross-reactivity supports the notion of structural and functional relatedness between the A and E molecules and suggests a common evolutionary origin of the A- and E-encoding loci.     

Ex vivo-expanded CD4+CD25+ immunoregulatory T cells prevent graft-versus-host-disease by inhibiting activation/differentiation of pathogenic T cells

CD4+CD25+ immunoregulatory T cells (Tregs) can be administered to inhibit graft-vs-host disease (GVHD) while preserving graft-vs-leukemia activity after allogeneic bone marrow transplantation in mice. Preclinical studies suggest that it is necessary to infuse as many Tregs as conventional donor T cells to achieve a clinical effect on GVHD. Thus, it would be necessary to expand Tregs ex vivo before transplantation. Two strategies have been proposed: expansion of Tregs stimulated by anti-CD3/CD28-coated microbeads for polyclonal activation or by host-type allogeneic APCs for selecting Tregs specific for host Ags. In this study, we describe the mechanisms by which ex vivo-expanded Tregs act on donor T cells to prevent GVHD in mice. We demonstrate that expanded Tregs strongly inhibited the division, expansion, and differentiation of donor T cells, with a more pronounced effect with Tregs specific for host Ags. These latter cells permit the efficient and durable control of GVHD and favor immune reconstitution.     

The origin and fate of beta 2m-free MHC class I molecules induced on activated T cells.

We report here that the expression of major histocompatibility complex (MHC) class I heavy chains not associated with beta 2-microglobulin is induced on resting human T cells by a variety of stimuli. These beta 2m-free class I heavy chains are not transported as such from the endoplasmic reticulum but originate from surface beta 2m-associated MHC class I molecules. beta 2m-free class I heavy chains are spontaneously released from the surface of activated cells. Cross-linking of beta 2m-free class I heavy chains with specific monoclonal antibodies results in the rapid down-regulation and internalization of these molecules. In contrast, beta 2m-associated MHC class I molecules display a different pattern of modulation. Previously, we reported that beta 2m-free class I heavy chains interact with CD8 molecules expressed on the same activated T cells. We propose that interactions between these molecules are involved in a mechanism regulating the function of activated T cells.     

Regulatory T cells control uveoretinitis induced by pathogenic Th1 cells reacting to a specific retinal neoantigen

In many clinical cases, uveitis develops secondary to an infection. This could result from peripheral activation followed by ocular penetration and reactivation of T cells specific for microbial Ags expressed in the retina. To gain insights into the pathophysiology of uveitis, we developed a new mouse model based on stable retinal expression of influenza virus hemagglutinin (HA) neoantigen by adeno-associated virus-mediated gene transfer. One month thereafter, we adoptively transferred HA-specific T cells, which were activated in vitro or in vivo. Intraocular inflammation was clinically and histologically observed in all animals within 15 days. The ocular infiltrate was composed mostly of macrophages and HA-specific T cells with a proinflammatory cytokine profile. Depletion of CD4(+)CD25(+) regulatory T cells exacerbated the disease, whereas HA-specific CD4(+)CD25(+) T cells given i.v. controlled the disease. This novel model should allow to better study the pathophysiology and therapeutic of uveitis.     

Human lymphocyte activation gene-3 molecules expressed by activated T cells deliver costimulation signal for dendritic cell activation

Data have been reported on the in vivo adjuvant role of soluble lymphocyte activation gene-3 (LAG-3) recombinant protein in mouse models and on its ability to support the in vitro generation of human, tumor-specific CTLs. In this study, we show that soluble human rLAG-3 protein (hLAG-3Ig) used in vitro as a single maturation agent induces phenotypic maturation of monocyte-derived dendritic cells and promoted the production of chemokines and TNF-alpha inflammatory cytokine. When given in association with optimal or suboptimal doses of CD40/CD40L, hLAG-3Ig functions as a strong costimulatory factor and induces full functional activation of monocyte-derived dendritic cells that includes the production of high level of IL-12p70. Moreover, evidence is here provided that this costimulatory function licensing dendritic cells to produce IL-12p70 is also a functional property of LAG-3 molecules when expressed in a physiological context by CD4(+) activated T cells. Altogether, these data show for the first time a role of LAG-3 in mediating dendritic cell activation when expressed on the T cell surface or released after specific Ag stimulation in the interspaces of immunological synapses.     

Immunoglobulin on activated T cells detected by indirect immunofluorescence

A high proportion of H2 antigen-activated thymus-derived thoracic duct lymphocytes stained positively with rabbit anti-mouse immunoglobulin reagents as detected by indirect immunofluorescence. In view of the specificity of the reagents used and the fact that T cells from other sources e.g., thymus, were not stained by this technique, it was concluded that the material detected on H-2 antigen-activated thymus-derived thoracic duct lymphocytes was indeed immunoglobulin. When H-2 antigen-activated thymus-derived thoracic duct lymphocytes were cultured in vitro at 37 °C for 18 hr, with or without prior treatment with chymotrypsin, surface immunoglobulin could no longer be detected on the cells. This suggested that immunoglobulin had not been synthesized by the cells but absorbed from elsewhere.     

The augmentation of surface Ly-6A/E molecules in activated T cells is mediated by endogenous interferon-gamma

The murine Ly-6A.2 and Ly-6E.1 antigens, which can transduce triggering signals in T cells, have been shown to become highly expressed after mitogenic stimulation. It has recently been found that enhanced expression of Ly-6A/E antigens is also induced by interferon-gamma (IFN-gamma) in resting T cells. Here, the possibility is investigated that Ly-6A/E induction on activated T cells may be due to the IFN-gamma known to be secreted by these cells. A potent neutralizing anti-IFN-gamma monoclonal antibody (mAb) (H-22.10) was used. This mAb was found to abrogate the augmentation of Ly-6A/E antigens produced in resting T cells by supernatants from T cells stimulated with concanavalin A. When added directly into cultures of T cells stimulated with concanavalin A or by the combination of ionomycin with the protein kinase C activator phorbol myristate acetate (PMA), the H-22.10 mAb inhibited Ly-6A/E enhancement without affecting the blastogenesis or the emergence of interleukin 2 receptors and transferrin receptors. Such a selective effect of the anti-IFN-gamma mAb indicated that IFN-gamma is involved in the up-regulation of Ly-6A/E antigens during T cell activation. In determining whether other activation signals, in addition to IFN-gamma receptor occupancy, may contribute to Ly-6A/E enhancement, it was found that suboptimal stimulation of BALB/c T cells provided by a 3-hr pulse with ionomycin plus PMA or by culture with PMA alone potentiated by about twofold the increase of Ly-6E.1 induced by exogenous IFN-gamma. Therefore, Ly-6A/E augmentation in activated T cells reflects primarily an action of endogenous IFN-gamma that is amplified (in BALB/c mice) by a protein kinase C-dependent step.     

Tumor cells prevent mouse dendritic cell maturation induced by TLR ligands

Tumor cells can evade the immune system through several mechanisms, one of which is to block DC maturation. It has been suggested that signaling via Toll-like receptors (TLR) may be involved in the induction of prophylactic anti-cancer immunity and in the treatment of established tumors. In the present study we found that high numbers of tumor cells interfere with BMDC activation induced by the TLR ligands LPS and poly IC. Tumor cells blocked TLR3- and TLR4-mediated induction of MHCII and the co-stimulatory molecules CD40 and CD86, as well as the cytokines IL-12, TNF-α and IL-6. Importantly, tumor cells induced inhibitory molecules (B7-DC, B7-H1 and CD80) on spleen DC in vivo and on BMDC, even in the presence of TLR ligands. Moreover, after a long exposure with tumor cells, purified BMDC were unable to respond to a second challenge with TLR ligands. The failure of tumor exposed-BMDC to express co-stimulatory molecules and cytokines in the presence of TLR ligands has implications for the future development of DC-based cancer immune therapies using TLR ligands as adjuvants for the activation of DC.     

Autoantigen-specific TGFbeta-induced Foxp3+ regulatory T cells prevent autoimmunity by inhibiting dendritic cells from activating autoreactive T cells

Several strategies are being designed to test the therapeutic potential of Ag-specific regulatory T cells to prevent or treat autoimmune diseases. In this study, we demonstrate that naive CD4+ Foxp3- T cells specific for a naturally expressed autoantigen (H+/K+ ATPase) can be converted to Foxp3+ T regulatory cells (Tregs) when stimulated in presence of TGFbeta. TGFbeta-induced Tregs (iTregs) have all the characteristics of naturally generated regulatory T cells in vitro, and more importantly, are effective at preventing organ-specific autoimmunity in a murine model of autoimmune gastritis. H+/K+ ATPase specific iTregs were able to inhibit the initial priming and proliferation of autoreactive T cells, and appear to do so by acting on H+/K+ ATPase presenting dendritic cells (DC). DC exposed to iTregs in vivo were reduced in their ability to stimulate proliferation and cytokine production by H+/K+ ATPase specific T cells. iTregs specifically reduced CD80 and CD86 expression on the surface of H+/K+ ATPase presenting DC in vitro. These studies reveal the therapeutic potential of Ag specific iTregs to prevent autoimmunity, and provide a mechanism by which this population of regulatory T cells, and perhaps others, mediate their suppressive effects in vivo.     

Shiga toxins expressed by human pathogenic bacteria induce immune responses in host cells

Shiga toxins are a family of genetically and structurally related toxins that are the primary virulence factors produced by the bacterial pathogens Shigella dysenteriae serotype 1 and certain Escherichia coli strains. The toxins are multifunctional proteins inducing protein biosynthesis inhibition, ribotoxic and ER stress responses, apoptosis, autophagy, and inflammatory cytokine and chemokine production. The regulated induction of inflammatory responses is key to minimizing damage upon injury or pathogen-mediated infections, requiring the concerted activation of multiple signaling pathways to control cytokine/chemokine expression. Activation of host cell signaling cascades is essential for Shiga toxin-mediated proinflammatory responses and the contribution of the toxins to virulence. Many studies have been reported defining the inflammatory response to Shiga toxins in vivo and in vitro, including production and secretion of tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), macrophage inflammatory protein-1α/β (MIP-1α/β), macrophage chemoattractant monocyte chemoattractant protein 1 (MCP-1), interleukin 8 (IL-8), interleukin 6 (IL-6), and Groβ. These cytokines and chemokines may contribute to damage in the colon and development of life threatening conditions such as acute renal failure (hemolytic uremic syndrome) and neurological abnormalities. In this review, we summarize recent findings in Shiga toxin-mediated inflammatory responses by different types of cells in vitro and in animal models. Signaling pathways involved in the inflammatory responses are briefly reviewed.     

Regulatory T cells induced by B cells: a novel subpopulation of regulatory T cells

Regulatory T cells play a crucial role in the homeostasis of the immune response. In addition to CD4⁺Foxp3⁺ regulatory T cells, several subsets of Foxp3^- regulatory T cells, such as T helper 3 (Th3) cells and type 1 regulatory T (Tr1) cells, have been described in mice and human. Accumulating evidence shows that naïve B cells contribute to tolerance and are able to promote regulatory T cell differentiation. Naïve B cells can convert CD4⁺CD25^- T cells into CD25⁺Foxp3^- regulatory T cells, named Treg-of-B cells by our group. Treg-of-B cells express LAG3, ICOS, GITR, OX40, PD1, and CTLA4 and secrete IL-10. Intriguingly, B-T cell-cell contact but not IL-10 is essential for Treg-of-B cells induction. Moreover, Treg-of-B cells possess both IL-10-dependent and IL-10-independent inhibitory functions. Treg-of-B cells exert suppressive activities in antigen-specific and non-antigen-specific manners in vitro and in vivo. Here, we review the phenotype and function of Foxp3⁺ regulatory T cells, Th3 cells, Tr1 cells, and Treg-of-B cells.     

Macrophage-derived nitric oxide inhibits the proliferation of activated T helper cells and is induced during antigenic stimulation of resting T cells

To examine how macrophage-derived nitric oxide (NO) affects T helper (Th) cell activity, T cell clones representing Th1 and Th2 subsets were activated before exposure to stimulated peritoneal macrophages or microglia. Both Th subsets were similarly sensitive to inhibition by NO, indicating that macrophage-derived NO regulates the proliferation of activated Th1 and Th2 cells equally well. Since IFN-gamma production remained intact in NO-treated Th1 cells, we studied whether NO was produced during antigen-specific activation of Th1 cells by unstimulated macrophages. Indeed, T cell proliferation only occurred when a NO synthase inhibitor was included, while IFN-gamma was essential for the induction of NO. These studies demonstrate that macrophages produce NO following antigen presentation to Th1 cells and that macrophage-derived NO inhibits Th1 and Th2 cell proliferation without inhibiting cytokine production.     

CD4 molecules are associated with the antigen receptor complex on activated but not resting T cells

To explore the relationship between CD4 and CD3/Ti on the T cell surface, we have studied a panel of Ag-specific Th cell lines and clones, as well as resting and mitogen-activated CD4+ cells. Our results show that exposure of Th cells to their specific antigenic stimuli, but not to irrelevant stimuli, induced the rapid disappearance of approximately 20 to 35% of CD3 and CD4 molecules. The modulation of these molecules was detected in less than 1 h, became maximal at 12 h, and recovered thereafter in parallel. Treatment of Th cells with anti-CD4 antibody prevented Ag-induced modulation of CD3, and treatment with anti-CD3 blocked modulation of CD4. In the absence of Ag, treatment of these cells with an antibody (WT-31) directed at a conformational determinant within CD3/Ti or with the combination of anti-CD3 antibody and goat anti-mouse Ig, also resulted in significant modulation of CD4. Similar treatment of PHA-activated CD4+ T cells with anti-CD3/Ti antibodies also induced CD4 modulation; however, the same antibodies failed to affect CD4 expression on fresh resting T cells. These results indicate that on activated, but not resting T cells, CD4 molecules can be physically associated with CD3/Ti. We postulate that this association is essential for efficient Th cell activation, and further that the ability of anti-CD4 antibodies to inhibit helper functions is due to their prevention of CD4-CD3/Ti interaction on the T cell surface.     

Regulatory T cells prevent CD8 T cell maturation by inhibiting CD4 Th cells at tumor sites

Natural regulatory T cells (Tregs) are present in high frequencies among tumor-infiltrating lymphocytes and in draining lymph nodes, supposedly facilitating tumor development. To investigate their role in controlling local immune responses, we analyzed intratumoral T cell accumulation and function in the presence or absence of Tregs. Tumors that grew in normal BALB/c mice injected with the 4T1 tumor cell line were highly infiltrated by Tregs, CD4 and CD8 cells, all having unique characteristics. Most infiltrating Tregs expressed low levels of CD25Rs and Foxp3. They did not proliferate even in the presence of IL-2 but maintained a strong suppressor activity. CD4 T cells were profoundly anergic and CD8 T cell proliferation and cytotoxicity were severely impaired. Depletion of Tregs modified the characteristics of tumor infiltrates. Tumors were initially invaded by activated CD4(+)CD25(-) T cells, which produced IL-2 and IFN-gamma. This was followed by the recruitment of highly cytotoxic CD8(+) T cells at tumor sites leading to tumor rejection. The beneficial effect of Treg depletion in tumor regression was abrogated when CD4 helper cells were also depleted. These findings indicate that the massive infiltration of tumors by Tregs prevents the development of a successful helper response. The Tregs in our model prevent Th cell activation and subsequent development of efficient CD8 T cell activity required for the control of tumor growth.     

Unresponsiveness of peripheral T cells induced by apoptotic bodies derived from autologous T cells

Several reports described the dose-dependent effect of Staphylococcus aureus enterotoxin B (SEB) regarding both levels of apoptosis and anergy of T cells. We investigated here whether T-cell apoptosis induced with SEB causes unresponsiveness of naive T cells. Apoptotic bodies were isolated from human T cells stimulated with antigen-presenting cells (APCs) and SEB by the continuous density gradient centrifugation method. When naive T cells were stimulated with APCs and SEB in the presence of apoptotic bodies, their proliferation was dose dependently suppressed and their TCRs were less downregulated than those of T cells stimulated without apoptotic bodies. Furthermore, those T cells were predisposed not to respond to restimulation with fresh APCs and SEB in the absence of apoptotic bodies. These results, taken together with the observation of tight binding of apoptotic bodies to APCs, imply that T cells stimulated in the presence of apoptotic bodies may undergo unresponsiveness due to interruption of contact with APCs.