Self major histocompatibility complex class I antigens expressed solely in lymphoid cells do not induce tolerance in the CD4+ T cell compartment.

Transgenic mice expressing self major histocompatibility complex (MHC) class I (H-2Kb) antigen solely in lymphoid cell lineages do not acquire tolerance to H-2Kb expressed on skin grafts. H-2Kb-specific cytotoxic T cell responses were completely abrogated in these mice, even after they had rejected skin grafts. Moreover, thymocytes expressing T cell receptors that confer H-2Kb reactivity on cytotoxic CD8+ T cells were eliminated. The ability to reject grafts correlated with the presence of a novel population of H-2Kb-reactive CD4+ T cells. At least some of these CD4+ T cells recognize peptides derived from H-2Kb by processing. We conclude that self MHC I antigens induce tolerance in the CD8 T cell compartment via negative selection when expressed exclusively by lymphoid cells. In contrast, tolerance to MHC class II-restricted self peptides derived by processing of such MHC I antigens is not induced in the CD4 T cell compartment. This suggests that effective transfer of self antigens from lymphoid cells to MHC II-positive cells that can process and present them as self peptides to thymocytes or CD4+ T cells does not take place in vivo. Thus, sequestration of self antigens and MHC II molecules in distinct cell types in the thymic microenvironment allows potentially autoreactive and functionally competent CD4+ T cells that recognize cryptic MHC II-restricted self peptides to mature into the peripheral T cell repertoire under normal physiological circumstances.

Eosinophilia in transgenic mice expressing interleukin 5.

Experiments in vitro suggest that although interleukin 5 (IL-5) stimulates the late stages of eosinophil differentiation, other cytokines are required for the generation of eosinophil progenitor cells. In this study transgenic mice constitutively expressing the IL-5 gene were established using a genomic fragment of the IL-5 gene coupled to the dominant control region from the gene encoding human CD2. Four independent eosinophilic transgenic lines have thus far been established, two of which with 8 and 49 transgene copies, are described in detail. These mice appeared macroscopically normal apart from splenomegaly. Eosinophils were at least 65- and 265-fold higher in blood from transgenics, relative to normal littermates, and approximately two- or sevenfold more numerous relative to blood from mice infected with the helminth Mesocestoides corti. Much more modest increases in blood neutrophil, lymphocyte, and monocyte numbers were noted in transgenics, relative to normal littermates (less than threefold). Thus IL-5 in vivo is relatively specific for the eosinophil lineage. Large numbers of eosinophils were present in spleen, bone marrow, and peritoneal exudate, and were highest in the line with the greatest transgene copy number. Eosinophilia was also noted in histological sections of transgenic lungs, Peyer's patches, mesenteric lymph nodes, and gut lamina propria but not in other tissues examined. IL-5 was detected in the sera of transgenics at levels comparable to those seen in sera from parasite-infected animals. IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) were not found. IL-5 mRNA was detected in transgenic thymus, Peyer's patches, and superficial lymph nodes, but not in heart, liver, brain, or skeletal muscle or in any tissues from nontransgenics. Bone marrow from transgenic mice was rich in IL-5-dependent eosinophil precursors. These data indicate that induction of the IL-5 gene is sufficient for production of eosinophilia, and that IL-5 can induce the full pathway of eosinophil differentiation. IL-5 may therefore not be restricted in action to the later stages of eosinophil differentiation, as suggested by earlier in vitro studies.

Inhibition of T cell proliferation by macrophage tryptophan catabolism.

We have recently shown that expression of the enzyme indoleamine 2, 3-dioxygenase (IDO) during murine pregnancy is required to prevent rejection of the allogeneic fetus by maternal T cells. In addition to their role in pregnancy, IDO-expressing cells are widely distributed in primary and secondary lymphoid organs. Here we show that monocytes that have differentiated under the influence of macrophage colony-stimulating factor acquire the ability to suppress T cell proliferation in vitro via rapid and selective degradation of tryptophan by IDO. IDO was induced in macrophages by a synergistic combination of the T cell-derived signals IFN-gamma and CD40-ligand. Inhibition of IDO with the 1-methyl analogue of tryptophan prevented macrophage-mediated suppression. Purified T cells activated under tryptophan-deficient conditions were able to synthesize protein, enter the cell cycle, and progress normally through the initial stages of G1, including upregulation of IL-2 receptor and synthesis of IL-2. However, in the absence of tryptophan, cell cycle progression halted at a mid-G1 arrest point. Restoration of tryptophan to arrested cells was not sufficient to allow further cell cycle progression nor was costimulation via CD28. T cells could exit the arrested state only if a second round of T cell receptor signaling was provided in the presence of tryptophan. These data reveal a novel mechanism by which antigen-presenting cells can regulate T cell activation via tryptophan catabolism. We speculate that expression of IDO by certain antigen presenting cells in vivo allows them to suppress unwanted T cell responses.

Levels of peripheral T cell tolerance induced by different doses of tolerogen.

Antigen-specific immunosuppression requires an understanding of the parameters that control peripheral T cell tolerance. A liver-specific inducible promoter was used to drive the expression of the major histocompatibility complex antigen Kb in transgenic mice. Minute amounts of Kb, expressed exclusively on hepatocytes, induced tolerance by partial down-regulation of the T cell receptor (TCR) on the self-reactive CD8+ cells. Contact of these tolerant T cells with high concentrations of Kb after induction led to complete down-regulation of TCR. Thus, tolerant T cells are susceptible to further tolerogenic signals and reach different levels of tolerance depending on antigen dose.

Prevention of allogeneic fetal rejection by tryptophan catabolism.

In 1953 Medawar pointed out that survival of the genetically disparate (allogeneic) mammalian conceptus contradicts the laws of tissue transplantation. Rapid T cell-induced rejection of all allogeneic concepti occurred when pregnant mice were treated with a pharmacologic inhibitor of indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme expressed by trophoblasts and macrophages. Thus, by catabolizing tryptophan, the mammalian conceptus suppresses T cell activity and defends itself against rejection.

Interaction between Kb and Q4 gene sequences generates the Kbm6 mutation.

Genetic interaction as a mechanism for the generation of mutations is suggested by recurrent, multiple nucleotide substitutions that are identical to nucleotide sequences elsewhere in the genome. We have sequenced the mutant K gene from the bm6 mouse, which is one of a series of eight closely related, yet independently occurring mutants known collectively as the "bg series." Two changes from the Kb gene are found, positioned 15 nucleotides apart: an A-to-T change and a T-to-C change in the codons corresponding to amino acids 116 and 121, resulting in Tyr-to-Phe and Cys-to-Arg substitutions, respectively. Hybridization analysis with an oligonucleotide specific for the altered Kbm6 sequence identifies one donor gene, Q4, located in the Qa region of the H-2 complex. The two altered nucleotides that differentiate Kbm6 and Kb are present in Q4 in a region where Kb and Q4 are otherwise identical for 95 nucleotides, delineating the maximum genetic transfer between the two genes. Because the Kbm6 mutation arose in an homozygous mouse these data indicate that the Q4 gene contains the only donor sequence and demonstrates that Q-region gene sequences can interact with the Kb gene to generate variant K molecules.

Acceptance of skin grafts between mice bearing different allelic forms of beta 2-microglobulin.

Single amino acid disparities in MHC class I molecules can elicit transplantation responses. Since beta 2 microglobulin (beta 2m) is noncovalently associated with class I antigens on the cell membrane we investigated whether the single amino acid polymorphism at position 85 (Asp-->Ala) in the mouse beta 2m molecule can cause skin graft rejection. A B2mb transgene was introduced into CBA(B2ma) mice which subsequently expressed both forms of beta 2m. Skin from these CBA beta 2mb transgenic mice was not rejected by the parental CBA strain. Previous studies showed that cytotoxic T lymphocyte (CTL) responses directed against beta 2mb use H2Kb as a restriction element. We therefore produced mice expressing H2Kb and H2Ab as well as beta 2mb by crossing CBA.beta 2mb mice with either CBA.Kb (CBK) transgenic mice or C3H.SW mice and used these as skin graft donors for beta 2mb negative littermates. In both cases rejection of transgenic skin only occurred when mice had received both a beta 2mb graft and an H2-disparate allograft lying adjacent in the same site. Introduction of the male specific antigen, H-Y, as a helper determinant did not result in rejection of beta 2mb skin. Neither did two CTL determinants (P91A and beta 2mb) on the same graft complement one another to elicit a transplantation response. Prior immunisation with tissues expressing the beta 2m disparity alone did not generate in vivo or in vitro beta 2mb-specific CTL responses, suggesting that this single amino acid difference is not sufficient to elicit a CTL or helper T cell response.

Expanded cohorts of maternal CD8+ T-cells specific for paternal MHC class I accumulate during pregnancy.

A recombinant H-2Kb transgene, GK, containing the human HLA-G gene promoter is expressed throughout the trophoblast when inherited paternally. Male GK transgenic mice were mated with female T-cell receptor (TCR) transgenic mice to assess the effect of fetal H-2Kb expression on maternal H-2Kb-specific CD8+ T-cells during pregnancy. The number of maternal H-2Kb-specific CD8+ T-cells in spleen increased significantly (approximately 3-fold) 10 days post coitus when the GK transgene was inherited from the father. A smaller (approximately 2-fold) increase was observed in the spleen of pregnant females mated with C57BL/10 (H-2b) males. No increase was observed in mothers mated to syngeneic male mice. In both cases where expanded cohorts of maternal CD8+ T-cells were observed the amount of surface CD8 and to a lesser extent, TCR molecules was reduced. No change in the amount of surface CD44 or CD45RB was detected when levels were compared with naive T-cells from control virgin female mice. Expanded cohorts of CD8+ T-cells were also detected in para-aortic and inguinal lymph nodes draining the uterus but no changes were observed in mesenteric lymph nodes. This study concludes that maternal CD8+ T-cells are exposed to paternally inherited fetal MHC class I antigens during pregnancy. Moreover, the phenotype of the CD8+ T-cells in maternal spleen and lymph nodes that drain the uterus is not typical of activated, antigen-experienced T-cells suggesting that contact with fetal H-2Kb molecules induces a state of functional unresponsiveness.

Tryptophan catabolism prevents maternal T cells from activating lethal anti-fetal immune responses.

The murine conceptus is protected from maternal immunity by cells expressing indoleamine dioxygenase (IDO), which catabolizes tryptophan. Induction of lethal maternal anti-fetal immunity requires effective pharmacologic inhibition of IDO enzyme activity and the presence of maternal T cells, but not B cells and also depends on the degree of maternal-fetal tissue incompatibility. Based on these findings, we propose a model to explain the role of IDO in suppressing maternal immunity and the mechanism of fetal allograft rejection, when IDO activity is inhibited during gestation. This model incorporates observations that fetal allograft rejection is T cell dependent, antibody-independent and is accompanied by a novel type of inflammation involving extensive complement deposition at the maternal-fetal interface, when IDO activity is blocked during murine pregnancy.

Regulation of prostaglandin synthesis and cell adhesion by a tryptophan catabolizing enzyme.

BACKGROUND: The tryptophan catabolizing enzyme, indoleamine 2,3, dioxygenase (IDO) is one of two mammalian enzymes, which can catabolize the rarest essential amino acid, tryptophan. IDO is inducible by cytokines such as interferon-gamma and plays a role in inflammation and maternal tolerance of fetal allografts, although its exact mode of action is unclear. Therefore, we investigated the circumstances under which IDO is expressed in vitro together with the effects of overexpression of IDO on the growth and morphology of cells. RESULTS: Overexpression of IDO in the murine macrophage cell line RAW 264.7 and the murine fibrosarcoma cell line MC57, resulted in the growth of macroscopic cell foci, with altered cell adhesion properties. The expression of IDO was also detected during adhesion of wild type, nontransfected cells in tissue culture to standard cell growth substrates. Inhibition of this expression, likewise resulted in alterations in cell adhesion. Overexpression of IDO or inhibition of endogenous IDO expression was accompanied by changes in metalloproteinase expression and also in the expression and activity of the cyclooxygenase enzymes. In the case of RAW cells, IDO effects on cell growth could be reversed by adding back prostaglandins. CONCLUSIONS: These results suggest that catabolism of the rarest essential amino acid may regulate processes such as cell adhesion and prostaglandin synthesis.

Meeting report of the first conference of the International Placenta Stem Cell Society (IPLASS).

The International Placenta Stem Cell Society (IPLASS) was founded in June 2010. Its goal is to serve as a network for advancing research and clinical applications of stem/progenitor cells isolated from human term placental tissues, including the amnio-chorionic fetal membranes and Wharton's jelly. The commitment of the Society to champion placenta as a stem cell source was realized with the inaugural meeting of IPLASS held in Brescia, Italy, in October 2010. Officially designated as an EMBO-endorsed scientific activity, international experts in the field gathered for a 3-day meeting, which commenced with "Meet with the experts" sessions, IPLASS member and board meetings, and welcome remarks by Dr. Ornella Parolini, President of IPLASS. The evening's highlight was a keynote plenary lecture by Dr. Diana Bianchi. The subsequent scientific program consisted of morning and afternoon oral and poster presentations, followed by social events. Both provided many opportunities for intellectual exchange among the 120 multi-national participants. This allowed a methodical and deliberate evaluation of the status of placental cells in research in regenerative and reparative medicine. The meeting concluded with Dr. Parolini summarizing the meeting's highlights. This further prepared the fertile ground on which to build the promising potential of placental cell research. The second IPLASS meeting will take place in September 2012 in Vienna, Austria. This meeting report summarizes the thought-provoking lectures delivered at the first meeting of IPLASS.

A key in vivo antitumor mechanism of action of natural product-based brassinins is inhibition of indoleamine 2,3-dioxygenase.

Agents that interfere with tumoral immune tolerance may be useful to prevent or treat cancer. Brassinin is a phytoalexin, a class of natural products derived from plants that includes the widely known compound resveratrol. Brassinin has been demonstrated to have chemopreventive activity in preclinical models but the mechanisms underlying its anticancer properties are unknown. Here, we show that brassinin and a synthetic derivative 5-bromo-brassinin (5-Br-brassinin) are bioavailable inhibitors of indoleamine 2,3-dioxygenase (IDO), a pro-toleragenic enzyme that drives immune escape in cancer. Like other known IDO inhibitors, both of these compounds combined with chemotherapy to elicit regression of autochthonous mammary gland tumors in MMTV-Neu mice. Furthermore, growth of highly aggressive melanoma isograft tumors was suppressed by single agent treatment with 5-Br-brassinin. This response to treatment was lost in athymic mice, indicating a requirement for active host T-cell immunity, and in IDO-null knockout mice, providing direct genetic evidence that IDO inhibition is essential to the antitumor mechanism of action of 5-Br-brassinin. The natural product brassinin thus provides the structural basis for a new class of compounds with in vivo anticancer activity that is mediated through the inhibition of IDO.

Analysis of major histocompatibility complex class I gene transcription using oligonucleotide probes.

Many highly homologous genes are present in the murine major histocompatibility complex (MHC) class I gene family. Consequently, it is difficult to distinguish between RNA transcripts of individual class I genes solely on the basis of nucleic acid hybridization analysis using DNA probes over 50 base pairs long. To avoid this problem, I have designed and synthesized a set of oligonucleotide probes capable of detecting transcripts of single class I genes in the MHC of C57BL/10 mice or sets of allelic class I genes at the same genetic locus in MHC disparate mouse strains. Using these probes, it is possible to determine the relative abundance of specific class I gene transcripts in a wide variety of cell and tissue types from inbred or MHC disparate mice. Examples of the use of these probes to detect different class I gene transcripts in cloned murine T cells, T cells transformed with Radiation Leukemia Virus, chemically induced thymoma cell lines and embryonic tissues are described. The results of these experiments are discussed in the light of possible roles of class I antigens in tumorigenesis or in early development.

Tolerance and MHC restriction in transgenic mice expressing a MHC class I gene in erythroid cells.

Transgenic mice carrying a MHC class I structural gene (H-2Kb) linked to transcriptional control elements from the human beta-globin gene, which direct erythroid lineage specific transcription, express H-2Kb molecules in red blood cells but H-2Kb expression cannot be detected in skin or lymphoid cells. This limited pattern of self MHC expression is sufficient to induce tolerance to H-2Kb molecules and H-2Kb restricted cytotoxic T cell responses can be generated in transgenic mice. Transgenic mice are unable to mount H-2Kb specific cytotoxic T cell responses in vitro, even when exogenous IL-2 is provided. However, H-2Kb specific T cell proliferative responses are comparable with H-2Kb specific responses in non-transgenic mice, even in the absence of exogenous IL-2. Thus, expression of H-2Kb molecules under control of human beta-globin transcriptional control elements in transgenic mice is tolerogenic but does not result in elimination of all H-2Kb reactive T cells from the mature repertoire. This suggests that tolerance in these mice may arise due to functional inactivation of H-2Kb reactive T cells in vivo when they encounter H-2Kb molecules expressed on cells of erythroid cell lineages or on non-erythroid cells which express H-2Kb molecules at very low levels or in a developmentally regulated pattern. Furthermore, in spite of the failure to detect H-2Kb expression on non-erythroid cells in these mice, we conclude that H-2Kb molecules participate in positive selection of the T cell repertoire since H-2Kb restricted T cell responses can be generated in these transgenic mice.

Immunology at the maternal-fetal interface: lessons for T cell tolerance and suppression.

Mammalian reproduction poses an immunological paradox because fetal alloantigens encoded by genes inherited from the father should provoke responses by maternal T cells leading to fetal loss. Current understanding of T cell immunobiology and the critical role of inflammatory processes during pregnancy is reviewed and discussed. Lessons derived from studies on the regulation of T cell responsiveness during mammalian gestation are considered in the wider context of T cell tolerance toward some microbial infections and tumors, avoidance of autoimmunity, and tissue allograft rejection.

Extinguishing maternal immune responses during pregnancy: implications for immunosuppression.

Mammals owe their existence to immunosuppressive processes that prevent fetal rejection in utero. Blocking tryptophan catabolism during murine pregnancy allows maternal T cells to provoke fetal allograft rejection. Cells expressing indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan, prevent T cell cycle progression and enhance activation induced T cell death. Here, we discuss the role of cells expressing IDO in regulating maternal T cell immunity during pregnancy and consider whether this mechanism might contribute to immunological discrimination by promoting T cell tolerance in other circumstances.

Tryptophan catabolism and T-cell tolerance: immunosuppression by starvation?

Some macrophages inhibit microbial infections by producing indoleamine 2,3 dioxygenase (IDO), which catabolizes tryptophan. Here, Andrew Mellor and David Munn discuss evidence that cells that synthesize IDO protect the mammalian fetus from maternal T-cell attack and argue that this mechanism might have wider implications for the control of T-cell responses.