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T cell responses to symbionts in the intestine drive tolerance or inflammation depending on the genetic background of the host. These symbionts in the gut sense the available nutrients and adapt their metabolic programs to use these nutrients efficiently. Here, we ask whether diet can alter the expression of a bacterial antigen to modulate adaptive immune responses. We generated a CD4+ T cell hybridoma, BθOM, specific for Bacteroides thetaiotaomicron (B. theta). Adoptively transferred transgenic T cells expressing the BθOM TCR proliferated in the colon, colon-draining lymph node, and spleen in B. theta-colonized healthy mice and differentiated into regulatory T cells (Tregs) and effector T cells (Teffs). Depletion of B. theta-specific Tregs resulted in colitis, showing that a single protein expressed by B. theta can drive differentiation of Tregs that self-regulate Teffs to prevent disease. We found that BθOM T cells recognized a peptide derived from a single B. theta protein, BT4295, whose expression is regulated by nutrients, with glucose being a strong catabolite repressor. Mice fed a high-glucose diet had a greatly reduced activation of BθOM T cells in the colon. These studies establish that the immune response to specific bacterial antigens can be modified by changes in the diet by altering antigen expression in the microbe.

Summary Microbes interact with the host immune system via several potential mechanisms. One essential step for each mechanism is the method by which intestinal microbes or their antigens access specific host immune cells. Using genetically susceptible mice ( dnKO ) that develop spontaneous, fulminant colitis, triggered by Bacteroides thetaiotaomicron ( B. theta ), we investigated the mechanism of intestinal microbial access under conditions that stimulate colonic inflammation. B. theta antigens localized to host immune cells through outer membrane vesicles (OMVs) that harbor bacterial sulfatase activity. We deleted the anaerobic sulfatase maturating enzyme (anSME) from B. theta , which is required for post-translational activation of all B. theta sulfatase enzymes. This bacterial mutant strain did not stimulate colitis in dnKO mice. Lastly, access of B. theta OMVs to host immune cells was sulfatase dependent. These data demonstrate that bacterial OMVs and associated enzymes promote inflammatory immune stimulation in genetically susceptible hosts.

Fetal surgery is a promising strategy to treat fetuses with severe congenital abnormalities but its clinical applications are often limited by preterm labor. In normal pregnancy, multiple mechanisms protect the semi-allogeneic fetus from attack by maternal T cells. Maternal microchimerism (the presence of maternal cells in the fetus) has been suggested to be one mechanism of maternal-fetal tolerance in that it exposes the fetus to non-inherited maternal antigens and leads to the generation of fetal regulatory T cells that can suppress a maternal T cell response. Preterm labor may represent a breakdown of this robust tolerance network. We hypothesized that during inflammation-associated preterm labor, maternal leukocytes cross the maternal-fetal interface and enter the fetal circulation. Consistent with this hypothesis, we found that during preterm labor in mice, the percentage of maternal microchimerism in fetal blood increased and the frequency of fetuses with high levels of trafficking (greater than 0.5%) also increased. Finally, we showed that the maternal leukocytes trafficking into the fetus are primarily Gr-1(+) cells in both syngeneic and allogeneic pregnancy, while T cell trafficking into the fetus specifically increases during allogeneic pregnancies. Our results demonstrate that trafficking of maternal leukocytes during pregnancy is altered during preterm labor. Such alterations may be clinically significant in affecting maternal-fetal tolerance.

Transplantation of allogeneic stem cells into the early gestational fetus, a treatment termed in utero hematopoietic cell transplantation (IUHCTx), could potentially overcome the limitations of bone marrow transplants, including graft rejection and the chronic immunosuppression required to prevent rejection. However, clinical use of IUHCTx has been hampered by poor engraftment, possibly due to a host immune response against the graft. Since the fetal immune system is relatively immature, we hypothesized that maternal cells trafficking into the fetus may pose the true barrier to effective IUHCTx. Here, we have demonstrated that there is macrochimerism of maternal leukocytes in the blood of unmanipulated mouse fetuses, with substantial increases in T cell trafficking after IUHCTx. To determine the contribution of these maternal lymphocytes to rejection after IUHCTx, we bred T and/or B cell-deficient mothers to wild-type fathers and performed allogeneic IUHCTx into the immunocompetent fetuses. There was a marked improvement in engraftment if the mother lacked T cells but not B cells, indicating that maternal T cells are the main barrier to engraftment. Furthermore, when the graft was matched to the mother, there was no difference in engraftment between syngeneic and allogeneic fetal recipients. Our study suggests that the clinical success of IUHCTx may be improved by transplanting cells matched to the mother.

Rhabdomyosarcoma is a childhood tumor of the skeletal muscle lineage in which cells display defects in both biochemical and morphological aspects of differentiation. The immunoglobulin superfamily members CDO and BOC are components of a cell surface receptor that positively regulates myogenesis in vitro. Expression of Cdo and Boc in myoblast cell lines is downregulated by the ras oncogene, and forced re-expression of either Cdo or Boc can override ras-induced inhibition of myogenic differentiation [Kang et al., J Cell Biol 1998; 143:403-413; Kang et al., EMBO J 2002; 21:114-124]. The current study sought to test whether the promyogenic properties of CDO and BOC could be extended to a human rhabdomyosarcoma cell line, RD. Stable overexpression of CDO or BOC in RD cells led to enhanced expression of two markers of muscle cell differentiation, troponin T and myosin heavy chain, and to increased formation of elongated, myosin heavy chain-positive myotubes. These observations are consistent with the notion that CDO and BOC play a role in the inverse relationship between differentiation and transformation of cells in the skeletal muscle lineage.

In utero hematopoietic cell transplantation (IUHCTx) is a promising strategy for the treatment of congenital stem cell disorders. Despite the purported immaturity of the fetal immune system, the clinical success of this strategy has been limited by poor engraftment of transplanted cells. The fetal host immune system is thought to be the major barrier to achieving successful IUHCTx. Since the fetal immune system is immature, however, we hypothesized that the maternal immune response may instead pose the true barrier to IUHCTx. We have demonstrated that maternal T cells traffic into the fetus after allogeneic in utero transplantation and that these lymphocytes play a critical role in limiting engraftment. Furthermore, we have shown that MHC matching the donor cells to the mother improves engraftment in the unmatched fetus. These results help renew interest in using the fetal environment to treat patients with congenital stem cell disorders.

Fetal interventions to diagnose and treat congenital anomalies are growing in popularity but often lead to preterm labor. The possible contribution of the maternal adaptive immune system to postsurgical pregnancy complications has not been explored. We recently showed that fetal intervention in mice increases maternal T cell trafficking into the fetus and hypothesized that this process also may lead to increased maternal T cell recognition of the foreign conceptus and subsequent breakdown in maternal-fetal tolerance. In this study, we show that fetal intervention in mice results in accumulation of maternal T cells in the uterus and that these activated cells can produce effector cytokines. In adoptive transfer experiments, maternal T cells specific for a fetal alloantigen proliferate after fetal intervention, escape apoptosis, and become enriched compared with endogenous T cells in the uterus and uterine-draining lymph nodes. Finally, we demonstrate that such activation and accumulation can have a functional consequence: in utero transplantation of hematopoietic cells carrying the fetal alloantigen leads to enhanced demise of semi-allogeneic fetuses within a litter. We further show that maternal T cells are necessary for this phenomenon. These results suggest that fetal intervention enhances maternal T cell recognition of the fetus and that T cell activation may be a culprit in postsurgical pregnancy complications. Our results have clinical implications for understanding and preventing complications associated with fetal surgery such as preterm labor. Copyright © 2014 by The American Association of Immunologists, Inc.

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency. In several other conditions, however, IUT has been attempted without success. Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice and those with genetic diseases. The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral, adeno-associated viral, retroviral, and lentiviral vectors into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein. In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment. The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

Background: In utero hematopoietic cell transplantation (IUHCTx) is a promising strategy to treat congenital diseases and induce donor-specific tolerance. The role of regulatory T cells (Tregs) in the establishment and maintenance of chimerism after IUHCTx has not been established. We characterized the expansion of Tregs in a wildtype (wt) and antigen-specific setting after IUHCTx. Methods: We analyzed chimerism in BALB/c fetuses 5 weeks after allogeneic IUHCTx and characterized their T cell and APC subsets. To quantify antigen-specific Tregs, BALB/c cells were transplanted into T cell receptor transgenic fetuses that recognize BALB/c antigen via the direct (4C) or indirect (TCR75) pathway. Results: In wt animals, we found an equivalent percentage of Tregs among uninjected animals, chimeras, and injected non-chimeras. The percentage of activated CD44+ Tregs was significantly higher among chimeras compared to uninjected controls. APC maturation markers (B7 and CD40) were elevated in donor and host-derived pDC and B cells of chimeric animals. In both 4C and TCR75 recipients, chimeric mice showed deletion of T effector cells and expansion of Tregs in the thymus and spleen. 4C animals losing chimerism showed a decrease in the number of Tregs. Conclusions: Our data suggests that Tregs interact with both host and donor-derived APC and undergo activation. Successful Treg induction is important for establishing and maintaining chimerism after IUHCTx.

Purpose: Multiple tolerance mechanisms protect the semi-allogeneic fetus from attack by the maternal adaptive immune system. We hypothesize preterm labor (PTL) may represent a breakdown of tolerance to the allogeneic fetus. Methods: B6 females were bred to BALB/c males and CFSE-labeled TCR transgenic T cells (TCR75 CD4, indirect; 4C CD4, direct; 2C CD8, direct) which recognize BALB/c antigen were adoptively transferred on E12.5 to study the roles of indirect and direct antigen presentation. PTL was induced by LPS on E13.5 and mice were analyzed at E18.5 for expression of CD44, CD62L, and CFSE. Controls included syngeneic matings with PTL and allogeneic matings without PTL. Results: 4C and 2C T cells did not proliferate during PTL. TCR75 cells proliferated and upregulated CD44 in allogeneic pregnancies with PTL compared to those without PTL (p

Background: Preterm labor (PTL) is a devastating consequence of fetal interventions such as in utero hematopoietic cell transplantation (IUHCTx). We previously found that PTL after allogeneic IUHCTx is decreased in mice lacking T cells. We hypothesized that the T cell profile in the uterine draining lymph nodes (uLN) would be altered in mothers with PTL. Methods: Mothers carrying semi-allogeneic fetuses underwent IUHCTx at E14.5. At E18.5, we quantified maternal CD4+, CD8+, and CD4+Foxp3+ T cells and measured the production of IL2, IL4, IFN gamma, and IL17 in the uLN, spleen, or non-draining lymph nodes (ndLN). Results: We found equivalent numbers of CD4+ and CD8+ T cells when comparing mothers with and without PTL. We observed a significant increase in the proportion of CD4+Foxp3+ T cells only in the uLN of mothers who experienced complete fetal loss. We also observed equivalent IL2, IFNγ, and IL17 production between all groups. IL4 production by CD4+Foxp3+ T cells was, however, significantly elevated in the uLN of mothers who aborted their entire litter. Conclusions: PTL after fetal intervention leads to expansion of the CD4+Foxp3+ T cell pool in the uLN. The production of IL4 by CD4+Foxp3+ T cells in the uLN suggests that the conversion of these cells to Th2 T effectors may be one potential mechanism by which maternal T cells participate in PTL. Our results support the role of maternal T cells in the pathogenesis of PTL.

Abstract 64 In utero hematopoietic stem cell transplantation (IUHSCTx) is a promising treatment strategy for many congenital hematopoietic disorders such as immunodeficiencies. However, clinical applications have been hampered by lack of engraftment, possibly secondary to a host immune response. This has been a conundrum in the field, since the fetus can also be tolerized to allogeneic cells in some circumstances. We hypothesized that it is the maternal immune response which limits engraftment of in utero transplanted cells. Methods: Fetal BALB/c mice at 14 days' gestation were transplanted with age-matched fetal liver (FL) cells (2.5 × 106 cells/fetus) from allogeneic C57B6 mice and levels of circulating donor cell chimerism were determined serially starting at 4 weeks after in utero transplantation. Rates of engraftment (number of chimeric pups/number of surviving pups) and levels of chimerism (donor CD45 cells/total CD45 cells) were compared to controls in which animals were transplanted with congenic cells (C57B6 (CD45.2) fetal hosts transplanted with C57B6 (CD45.1) FL). In order to determine the role of the maternal adaptive immune system, immunodeficient BALB/c.Rag−/− mothers (deficient in T and B cells) were bred to wild type BALB/c males, such that the fetuses (BALB/c.Rag+/−) would be immunocompetent. These fetuses were transplanted with C57B6 FL and rates of engraftment and levels of chimerism in these transplants were compared to those in wild type allogeneic transplants. In order to determine whether the maternal influence is caused by maternal lymphocytes trafficking into the fetus, C57B6 (CD45.2) females were bred to C57B6 (CD45.1) males, such that the fetal cells (CD45.1+/CD45.2+) could be distinguished from maternal cells (CD45.1−/CD45.2+). Fetal blood and tissues were examined for the presence of maternal cells by flow cytometry at various gestational ages. Results: The rate of engraftment after IUHSCTx in control animals transplanted with congenic cells was 14/16 (88%) and average levels of chimerism were 9.9±8.4%. In contrast, the rate of engraftment in wild-type BALB/c fetuses transplanted with allogeneic B6 cells was 11/25 (44%; p Conclusions: There is an adaptive immune response which limits early engraftment after in utero transplantation of allogeneic cells and leads to a gradual decline in levels of chimerism in engrafted animals. However, in the selective absence of maternal T and B cells, all fetuses transplanted with allogeneic FL cells show long-term, multilineage engraftment and demonstrate donor-specific tolerance. These results indicate that the maternal immune system plays a significant role in the success of fetal HSC transplantation. Cellular trafficking between the mother and fetus may be a mechanism by which maternal lymphocytes encounter cells transplanted into the fetus. Our findings have clinical implications in that the success of IUHSCTx may be improved by harvesting cells from the mother or HLA-matching cells to the mother. Disclosures: No relevant conflicts of interest to declare.