Oral delivery of Hyperimmune bovine serum antibodies against CS6-expressing enterotoxigenic Escherichia coli as a prophylactic against diarrhea.

BACKGROUND: . Oral administration of bovine antibodies active against enterotoxigenic Escherichia coli (ETEC) have demonstrated safety and efficacy against diarrhea in human challenge trials. The efficacy of bovine serum immunoglobulins (BSIgG) against recombinant colonization factor CS6 or whole cell ETEC strain B7A was assessed against challenge with the CS6-expressing B7A. METHODS: . This was a randomized, double-blind, placebo-controlled trial in which healthy adults received oral hyperimmune BSIgG anti-CS6, anti-B7A whole cell killed or non-hyperimmune BSIgG (placebo) in a 1:1:1 ratio then challenged with ETEC B7A. Two days pre-challenge, volunteers began a thrice daily, seven day course of immunoprophylaxis. On day 3, subjects received 1 × 1010 CFUs of B7A. Subjects were observed for safety and the primary endpoint of moderate-severe diarrhea (MSD). RESULTS: . A total of 59 volunteers received product and underwent ETEC challenge. The BSIgG products were well-tolerated across all subjects. Upon challenge, 14/20 (70%) placebo recipients developed MSD, compared to 12/19 (63%; p = .74) receiving anti-CS6 BSIgG and 7/20 (35%; p = .06) receiving anti-B7A BSIgG. Immune responses to the ETEC infection were modest across all groups. CONCLUSIONS: . Bovine-derived serum antibodies appear safe and well tolerated. Antibodies derived from cattle immunized with whole cell B7A provided 50% protection against MSD following B7A challenge; however, no protection was observed in subjects receiving serum antibodies targeting CS6. The lack of observed efficacy in this group may be due to low CS6 surface expression on B7A, the high dose challenge inoculum and/or the use of serum derived antibodies versus colostrum-derived antibodies.

Immunomodulation of experimental colitis: the role of NK1.1 liver lymphocytes and surrogate antigens--bystander effect.

The imbalance between Th1 pro-inflammatory and Th2 anti-inflammatory cytokine-producing cells plays a major role in the pathogenesis of inflammatory bowel disease (IBD). Induction of oral tolerance to colitis-extracted proteins was previously shown to down-regulate the anti-colon immune response, thereby alleviating experimental colitis. Immune bystander effect and liver-associated lymphocytes expressing the NK1.1 marker (NK1.1(+) LAL) have been suggested as being important in tolerance induction. The aims of the present study were to determine whether oral administration of inflammatory and non-inflammatory colon-extracted proteins of different species can induce peripheral immune tolerance and alleviate experimental colitis; and to examine the role of NK1.1(+) LAL in oral tolerance induction. Colitis was induced in C57/B6 mice by intracolonic instillation of trinitrobenzene sulphonic acid (TNBS). Mice received six oral doses of colonic proteins extracted from TNBS-colitis colonic wall, or normal colonic wall, from four different species. Standard clinical, macroscopic, and microscopic scores were used for colitis assessment. Serum interferon gamma (IFNgamma) and interleukin 10 (IL10) levels were measured by ELISA. To evaluate the role of NK1.1(+) LAL in maintaining the balance between immunogenic and tolerogenic subsets of cells, their cytotoxicity functions were tested in tolerized and non-tolerized-mice. The administration of mouse-derived colitis-extracted proteins, or of surrogate proteins extracted from normal mouse colon, or from rat or human inflammatory colons, was found to alleviate experimental colitis. Tolerized mice had less diarrhoea; showed a marked reduction of colonic ulceration, intestinal and peritoneal adhesions, wall thickness, and oedema; and demonstrated a significant improvement of all microscopic parameters for colitis. Induction of tolerance led to an increase in IL10 and a decrease in IFNgamma serum levels. NK1.1(+) LAL cytotoxicity function increased markedly in tolerized mice. In contrast, mice fed with proteins extracted from normal rat, rabbit, and human colon, or from rabbit inflammatory colon, developed severe colitis, with a marked increase in IFNgamma and a decrease in IL10 serum levels, and down-regulation of NK1.1(+) LAL function. This study has shown that oral tolerance can be induced in experimental colitis by means of the feeding of surrogate antigens; this alleviates experimental colitis. NK1.1(+) LAL cytotoxicity function is associated with peripheral tolerance induction and may help to maintain the Th1/Th2 immune balance.

Overexpression of suppressor of cytokine signaling-1 impairs pre-T-cell receptor-induced proliferation but not differentiation of immature thymocytes.

Cytokines play an essential role during early T-cell development. However, the mechanisms controlling cytokine signaling in developing thymocytes have not been elucidated. Cytokine receptor signaling can be modulated by suppressor of cytokine signaling-1 (SOCS-1), which acts as a negative regulator of Janus kinases. SOCS-1 is normally expressed throughout thymocyte development; however, retroviral-mediated overexpression of SOCS-1 in fetal liver-derived hematopoietic progenitors prevented their progression beyond the earliest stage of T-cell development. Further analysis revealed that SOCS-1 expression is transiently suppressed following pre-T-cell receptor (TCR) signaling. Moreover, constitutive expression of SOCS-1 abrogated pre-TCR- mediated expansion of immature thymocytes but did not interfere with differentiation. These findings reveal that SOCS-1 serves to regulate cytokine signaling at critical checkpoints during early T-cell development.

Opposite ability of pre-TCR and alpha beta TCR to induce apoptosis.

In early CD4(-)CD8(-) pro-thymocytes, signaling through the pre-TCR is crucial for survival and differentiation into CD4(+)CD8(+) cells. At this more mature stage, interactions between alphabetaTCR and self-Ag/MHC complexes in turn lead either to cell survival and differentiation (positive selection) or to cell death (negative selection). Intrinsic differences must therefore exist between pre-TCR signals in CD4(-)CD8(-) thymocytes and alphabetaTCR signals in CD4(+)CD8(+) cells, since only the latter can mediate a death signal. In this work, we directly compared the capability of pre-TCR and alphabetaTCR to induce apoptosis in a CD4(-)CD8(-) thymoma cell line following receptor cross-linking with mAbs. Cross-linking of alphabetaTCR triggered high levels of programmed cell death, mimicking the negative selection signal usually induced in CD4(+)CD8(+) thymocytes. In contrast, pre-TCR was very inefficient at inducing apoptosis upon cross-linking, despite similar levels of surface receptor expression. Importantly, inefficient apoptosis induction by the pre-TCR did not result from its weak association with TCRzeta chain, since TCRs containing alpha-pTalpha chimeric chains, binding weakly to TCRzeta, were still able to induce apoptosis. Although similar tyrosine phosphorylation and calcium influx were induced after either pre-TCR or alphabetaTCR cross-linking, the two pathways diverged at the level of Fas ligand induction. Among putative transcription factors involved in Fas ligand mRNA induction, Nur77 and NFAT transcriptional activities were readily induced after alphabetaTCR, but not pre-TCR, stimulation. Together, these results support the view that the structure of the pre-TCR and alphabetaTCR directly influences their apoptosis-inducing capabilities by activating distinct signaling pathways.

Competitive displacement of pT alpha by TCR-alpha during TCR assembly prevents surface coexpression of pre-TCR and alpha beta TCR.

During alphabeta T cell development, CD4(-)CD8(-) thymocytes first express pre-TCR (pTalpha/TCR-beta) before their differentiation to the CD4(+)CD8(+) stage. Positive selection of self-tolerant T cells is then determined by the alphabeta TCR expressed on CD4(+)CD8(+) thymocytes. Conceivably, an overlap in surface expression of these two receptors would interfere with the delicate balance of thymic selection. Therefore, a mechanism ensuring the sequential expression of pre-TCR and TCR must function during thymocyte development. In support of this notion, we demonstrate that expression of TCR-alpha by immature thymocytes terminates the surface expression of pre-TCR. Our results reveal that expression of TCR-alpha precludes the formation of pTalpha/TCR-beta dimers within the endoplasmic reticulum, leading to the displacement of pre-TCR from the cell surface. These findings illustrate a novel posttranslational mechanism for the regulation of pre-TCR expression, which may ensure that alphabeta TCR expression on thymocytes undergoing selection is not compromised by the expression of pre-TCR.

Identification of a novel pre-TCR isoform in which the accessibility of the TCR beta subunit is determined by occupancy of the 'missing' V domain of pre-T alpha.

We have identified a novel pre-TCR isoform that is structurally distinct from conventional pre-TCR complexes and whose TCR beta chains are inaccessible to anti-TCR beta antibodies. We term this pre-TCR isoform the MB (masked beta)-pre-TCR. Pre-T alpha (pT alpha) subunits of MB-pre-TCR complexes have a larger apparent mol. wt due to extensive modification with O:-linked carbohydrates; however, preventing addition of O-glycans does not restore antibody recognition of the TCR beta subunits of MB-pre-TCR complexes. Importantly, accessibility of TCR beta chains in MB-pre-TCR complexes is restored by filling in the 'missing' variable (V) domain of pT alpha with a V domain from TCR alpha. Moreover, the proportion of pre-TCR complexes in which the TCR beta subunits are accessible to anti-TCR beta antibody varies with the cellular context, suggesting that TCR beta accessibility is controlled by a trans-acting factor. The way in which this factor might control TCR beta accessibility as well as the physiologic relevance of TCR beta masking for pre-TCR function are discussed.

Thymic selection generates T cells expressing self-reactive TCRs in the absence of CD45.

The CD45 protein tyrosine phosphatase regulates Ag receptor signaling in T and B cells. In the absence of CD45, TCR coupling to downstream signaling cascades is profoundly reduced. Moreover, in CD45-null mice, the maturation of CD4+CD8+ thymocytes into CD4+CD8- or CD4-CD8+ thymocytes is severely impaired. These findings suggest that thymic selection may not proceed normally in CD45-null mice, and may be biased in favor of thymocytes expressing TCRs with strong reactivity toward self-MHC-peptide ligands to compensate for debilitated TCR signaling. To test this possibility, we purified peripheral T cells from CD45-null mice and fused them with the BWalpha-beta- thymoma to generate hybridomas expressing normal levels of TCR and CD45. The reactivity of these hybridomas to self or foreign MHC-peptide complexes was assessed by measuring the amount of IL-2 secreted upon stimulation with syngeneic or allogeneic splenocytes. A very high proportion (55%) of the hybridomas tested reacted against syngeneic APCs, indicating that the majority of T cells in CD45-null mice express TCRs with high avidity for self-MHC-peptide ligands, and are thus potentially autoreactive. Furthermore, a large proportion of TCRs selected in CD45-null mice (H-2b) were also shown to display reactivity toward closely related MHC-peptide complexes, such as H-2bm12. These results support the notion that modulating the strength of TCR-mediated signals can alter the outcome of thymic selection, and demonstrate that CD45, by molding the window of affinity/avidity for positive and negative selection, directly participates in the shaping of the T cell repertoire.

Enhancement of immune tolerance via induction of NK1.1 positive liver-associated-lymphocytes under immunosuppressive conditions.

BACKGROUND/AIMS: The liver was previously shown to play a critical role in oral tolerance induction. A subset of liver-associated-lymphocytes expressing NK1.1 marker (NK1.1+ LAL) have killing activities and it has been suggested that they play a role in immune modulation. FK506 is a powerful immunosuppressive agent affecting T-cell differentiation and function. The exact pathway involved in peripheral tolerance induction using this drug remains unknown. The aim of the present study was to determine the interaction between FK506 and NK1.1+ LAL in induction of peripheral immune tolerance in the experimental colitis model. METHODS: Colitis was induced in C57 mice by intracolonic instillation of trinitrobenzenesulfonic acid (TNBS). Mice received five oral doses of colonic proteins extracted from TNBS-colitis colonic wall with and without FK506 treatment. The effect of FK506 treatment on NK1.1+ LAL was tested by cell-sorting and cytotoxicity assay. Colitis was assessed by standard clinical, macroscopic and histologic scores. RESULTS: Both FK506 treatment and oral tolerance induced a significant increase in NK1.1+ LAL number and cytotoxicity function. FK506 treatment enhanced the effect of oral tolerance on amelioration of disease activity. Orally tolerized mice treated with FK506 had no mortality nor increase in body weight, and manifested significant improvement in disease macroscopic and microscopic scores. CONCLUSIONS: This study shows for the first time that immune tolerance induced by both oral administration of an antigen and by FK506 treatment may be mediated via enhancement of NK1.1+ LAL. This subset of lymphocytes may play an immunoregulatory role in immune tolerance induction.

Extracellular signal-regulated kinase (ERK) activation by the pre-T cell receptor in developing thymocytes in vivo.

The first checkpoint in T cell development occurs between the CD4(-)CD8(-) and CD4(+)CD8(+) stages and is associated with formation of the pre-T cell receptor (TCR). The signaling mechanisms that drive this progression remain largely unknown. Here, we show that extracellular signal-regulated kinases (ERKs)-1/2 are activated upon engagement of the pre-TCR. Using a novel experimental system, we demonstrate that expression of the pre-TCR by developing thymocytes induces ERK-1/2 activation within the thymus. In addition, the activation of this pre-TCR signaling cascade is mediated through Lck. These findings directly link pre-TCR complex formation with specific downstream signaling components in vivo.

The connecting peptide domain of pT alpha dictates weak association of the pre-T cell receptor with the TCR zeta subunit.

Signals delivered through the pre-TCR, a heterodimer of pT alpha and TCR beta chains, are crucial for the maturation and proliferation of immature alphabeta lineage thymocytes from the CD4- CD8- to the CD4+ CD8+ stage. To gain insight into the structural and functional properties of the pre-TCR, chimeric TCR alpha chains were generated by replacing domains of the alpha chain cytoplasmic, transmembrane and constant regions with homologous domains from the pT alpha chain. All chimeric TCR could be expressed stably at the cell surface and induce Ca2+ mobilization as well as phosphorylation of several protein substrates on tyrosine residues. However, chimeras wherein the connecting peptide of TCR alpha chain was substituted by the one from pT alpha, were weakly associated with the TCR zeta chain, showing that functional but not physical interactions were preserved in such chimeras. In contrast, introduction of the connecting peptide of TCR alpha in the pT alpha chain was insufficient to confer stable association with the TCR zeta chain. These results demonstrate that the inability of the pre-TCR to interact strongly with TCR zeta is attributable to amino acid residues present throughout the region comprised between the intrachain Cys and the transmembrane domain. It remains to be determined whether the weak physical interaction between the pre-TCR alphand the zeta2 homodimer prevents the activation of specific TCR zeta-dependent signaling pathways, and thus confers unique signaling properties upon the pre-TCR. In addition, this structural difference between the pT alpha/beta and alphabeta TCR might constitute a means to regulate the expression of these receptors at the surface of thymocytes, at different stages of their maturation.

Liver-associated lymphocytes expressing NK1.1 are essential for oral immune tolerance induction in a murine model.

Oral tolerance is the induction of immunological hyporesponsiveness towards orally administered antigens. Tolerance initiation involves induction of anti-inflammatory (Th2) lymphocytes, with downregulation of pro-inflammatory (Th1) lymphocytes. The liver was previously shown to play a critical role in oral tolerance induction. The aim of the present study was to test whether liver-associated-lymphocytes expressing the NK1.1 marker (NK1.1+ LAL) are substantial for induction of oral tolerance in an experimental colitis model. Colitis was induced in C57 mice by intracolonic instillation of trinitrobenzensulfonic acid (TNBS). Mice received five oral doses of colonic proteins extracted from TNBS-colitis colonic wall. Anti-NK1.1 monoclonal antibodies were injected before tolerance induction. Colitis was assessed by standard clinical, macroscopic, and microscopic scores. Serum IFN-gamma, TGF-beta1, and IL4 levels were measured by enzyme-linked immunosorbent assay. To evaluate the role of NK1.1+ LAL in keeping the balance between immunogenic and tolerogenic subsets of cells, we tested whether peripheral lymphocytes harvested from tolerized and NK1.1-depleted nontolerized mice can adoptively transfer the tolerance into naive irradiated rats. Depletion of NK1.1+ LAL prevented immune tolerance induction in the experimental colitis model. NK1.1+ LAL-depleted nontolerized mice, disclosed severe clinical, macroscopic, and microscopic parameters of colitis. These mice had significantly lower TGF-beta1, IL4, and higher IFN-gamma serum levels, and their lymphocytes failed to transfer the tolerance into naive animals. In contrast, the feeding of colitis-extracted proteins, without NK1.1+ LAL depletion, markedly alleviated the disease. Tolerized mice had higher IL4 and TGF-beta1 and lower IFN-gamma serum levels, and adoptive transfer of their suppressor splenocytes markedly alleviated colitis in naive recipients. NK1.1+ LAL plays a critical role in oral tolerance induction. Depletion of this subset of LAL prevents a shift from Th1 to a Th2 type of immune response, hindering the ability to induce immune tolerance.

CD4+ CD8+ thymocytes are preferentially induced to die following CD45 cross-linking, through a novel apoptotic pathway.

Ligation of the protein tyrosine phosphatase CD45 on both mature and immature T cells modulates the amplitude of TCR-mediated signals. In this work, we have evaluated the consequences of CD45 ligation on immature T cells, in the absence of TCR engagement. Cross-linking of CD45 on thymocytes by mAbs led to the induction of cellular death, characterized by a reduction in mitochondrial membrane potential (delta psi(m)), production of reactive oxygen species, loss in membrane asymmetry, exposure of phosphatidylserine residues, and incorporation of vital dyes. In sharp contrast to most stimuli causing thymocyte death, CD45 cross-linking did not lead to DNA degradation. Cell death was not blocked by Bcl-2 overexpression or treatment with caspase inhibitor. However, death was inhibited by the addition of scavengers of reactive oxygen species. We also established that susceptibility to CD45-mediated death is acquired during the transition of early CD4- CD8- TCR- T cell precursors into CD4+ CD8+ TCR- thymocytes and is increased with further acquisition of surface TCR on these cells. Moreover, mature thymocytes were much less sensitive to CD45 cross-linking than CD4+ CD8+ cells. We propose that during T cell development, CD45 ligation could induce the death of those immature thymocytes that do not fulfill the requirements for positive selection.