Motile invaded neutrophils in the small intestine of Toxoplasma gondii-infected mice reveal a potential mechanism for parasite spread.

Toxoplasma gondii infection occurs through the oral route, but we lack important information about how the parasite interacts with the host immune system in the intestine. We used two-photon laser-scanning microscopy in conjunction with a mouse model of oral T. gondii infection to address this issue. T. gondii established discrete foci of infection in the small intestine, eliciting the recruitment and transepithelial migration of neutrophils and inflammatory monocytes. Neutrophils accounted for a high proportion of actively invaded cells, and we provide evidence for a role for transmigrating neutrophils and other immune cells in the spread of T. gondii infection through the lumen of the intestine. Our data identify neutrophils as motile reservoirs of T. gondii infection and suggest a surprising retrograde pathway for parasite spread in the intestine.

Conserved and divergent aspects of human T-cell development and migration in humanized mice.

Humanized mice represent an important model to study the development and function of the human immune system. While it is known that mouse thymic stromal cells can support human T-cell development, the extent of interspecies cross-talk and the degree to which these systems recapitulate normal human T-cell development remain unclear. To address these questions, we compared conventional and non-conventional T-cell development in a neonatal chimera humanized mouse model with that seen in human fetal and neonatal thymus samples, and also examined the impact of a human HLA-A2 transgene expressed by the mouse stroma. Given that dynamic migration and cell-cell interactions are essential for T-cell differentiation, we also studied the intrathymic migration pattern of human thymocytes developing in a murine thymic environment. We found that both conventional T-cell development and intra-thymic migration patterns in humanized mice closely resemble human thymopoiesis. Additionally, we show that developing human thymocytes engage in short, serial interactions with other human hematopoietic-derived cells. However, non-conventional T-cell differentiation in humanized mice differed from both fetal and neonatal human thymopoiesis, including a marked deficiency of Foxp3(+) T-cell development. These data suggest that although the murine thymic microenvironment can support a number of aspects of human T-cell development, important differences remain, and additional human-specific factors may be required.

Tracking migration during human T cell development.

Specialized microenvironments within the thymus are comprised of unique cell types with distinct roles in directing the development of a diverse, functional, and self-tolerant T cell repertoire. As they differentiate, thymocytes transit through a number of developmental intermediates that are associated with unique localization and migration patterns. For example, during one particular developmental transition, immature thymocytes more than double in speed as they become mature T cells that are among the fastest cells in the body. This transition is associated with dramatic changes in the expression of chemokine receptors and their antagonists, cell adhesion molecules, and cytoskeletal components to direct the maturing thymocyte population from the cortex to medulla. Here we discuss the dynamic changes in behavior that occur throughout thymocyte development, and provide an overview of the cell-intrinsic and extrinsic mechanisms that regulate human thymocyte migration.

Homeostatic cytokines reciprocally modulate the emergence of prenatal effector PLZF+CD4+ T cells in humans.

The development of human prenatal adaptive immunity progresses faster than previously appreciated, with the emergence of memory CD4+ T cells alongside regulatory T cells by midgestation. We previously identified a prenatal specific population of promyelocytic leukemia zinc finger-positive (PLZF+) CD4+ T cells with heightened effector potential that were enriched in the developing intestine and accumulated in the cord blood of infants exposed to prenatal inflammation. However, the signals that drive their tissue distribution and effector maturation are unknown. Here, we define the transcriptional and functional heterogeneity of human prenatal PLZF+CD4+ T cells and identify the compartmentalization of T helper-like (Th-like) effector function across the small intestine (SI) and mesenteric lymph nodes (MLNs). IL-7 was more abundant in the SI relative to the MLNs and drove the preferential expansion of naive PLZF+CD4+ T cells via enhanced STAT5 and MEK/ERK signaling. Exposure to IL-7 was sufficient to induce the acquisition of CD45RO expression and rapid effector function in a subset of PLZF+CD4+ T cells, identifying a human analog of memory phenotype CD4+ T cells. Further, IL-7 modulated the differentiation of Th1- and Th17-like PLZF+CD4+ T cells and thus likely contributes to the anatomic compartmentalization of human prenatal CD4+ T cell effector function.

Heritable vaginal bacteria influence immune tolerance and relate to early-life markers of allergic sensitization in infancy.

Maternal asthma status, prenatal exposures, and infant gut microbiota perturbation are associated with heightened risk of atopy and asthma risk in childhood, observations hypothetically linked by intergenerational microbial transmission. Using maternal vaginal (n = 184) and paired infant stool (n = 172) samples, we identify four compositionally and functionally distinct Lactobacillus-dominated vaginal microbiota clusters (VCs) that relate to prenatal maternal health and exposures and infant serum immunoglobulin E (IgE) status at 1 year. Variance in bacteria shared between mother and infant pairs relate to VCs, maternal allergy/asthma status, and infant IgE levels. Heritable bacterial gene pathways associated with infant IgE include fatty acid synthesis and histamine and tryptophan degradation. In vitro, vertically transmitted Lactobacillus jensenii strains induce immunosuppressive phenotypes on human antigen-presenting cells. Murine supplementation with L. jensenii reduces lung eosinophils, neutrophilic expansion, and the proportion of interleukin-4 (IL-4)+ CD4+ T cells. Thus, bacterial and atopy heritability are intimately linked, suggesting a microbial component of intergenerational disease transmission.

Mechanisms of Fetal T Cell Tolerance and Immune Regulation.

The developing human fetus generates both tolerogenic and protective immune responses in response to the unique requirements of gestation. Thus, a successful human pregnancy depends on a fine balance between two opposing immunological forces: the semi-allogeneic fetus learns to tolerate both self- and maternal- antigens and, in parallel, develops protective immunity in preparation for birth. This critical window of immune development bridges prenatal immune tolerance with the need for postnatal environmental protection, resulting in a vulnerable neonatal period with heightened risk of infection. The fetal immune system is highly specialized to mediate this transition and thus serves a different function from that of the adult. Adaptive immune memory is already evident in the fetal intestine. Fetal T cells with pro-inflammatory potential are born in a tolerogenic environment and are tightly controlled by both cell-intrinsic and -extrinsic mechanisms, suggesting that compartmentalization and specialization, rather than immaturity, define the fetal immune system. Dysregulation of fetal tolerance generates an inflammatory response with deleterious effects to the pregnancy. This review aims to discuss the recent advances in our understanding of the cellular and molecular composition of fetal adaptive immunity and the mechanisms that govern T cell development and function. We also discuss the tolerance promoting environment that impacts fetal immunity and the consequences of its breakdown. A greater understanding of fetal mechanisms of immune activation and regulation has the potential to uncover novel paradigms of immune balance which may be leveraged to develop therapies for transplantation, autoimmune disease, and birth-associated inflammatory pathologies.

CD161 contributes to prenatal immune suppression of IFNγ-producing PLZF+ T cells.

BACKGROUND: While the human fetal immune system defaults to a program of tolerance, there is concurrent need for protective immunity to meet the antigenic challenges encountered after birth. Activation of T cells in utero is associated with the fetal inflammatory response with broad implications for the health of the fetus and of the pregnancy. However, the characteristics of the fetal effector T cells that contribute to this process are largely unknown. METHODS: We analyzed primary human fetal lymphoid and mucosal tissues and performed phenotypic, functional, and transcriptional analysis to identify T cells with pro-inflammatory potential. The frequency and function of fetal-specific effector T cells was assessed in the cord blood of infants with localized and systemic inflammatory pathologies and compared to healthy term controls. RESULTS: We identified a transcriptionally distinct population of CD4+ T cells characterized by expression of the transcription factor Promyelocytic Leukemia Zinc Finger (PLZF). PLZF+ CD4+ T cells were specifically enriched in the fetal intestine, possessed an effector memory phenotype, and rapidly produced pro-inflammatory cytokines. Engagement of the C-type lectin CD161 on these cells inhibited TCR-dependent production of IFNγ in a fetal-specific manner. IFNγ-producing PLZF+ CD4+ T cells were enriched in the cord blood of infants with gastroschisis, a natural model of chronic inflammation originating from the intestine, as well as in preterm birth, suggesting these cells contribute to fetal systemic immune activation. CONCLUSION: Our work reveals a fetal-specific program of protective immunity whose dysregulation is associated with fetal and neonatal inflammatory pathologies.

Studying T Cell Development in Thymic Slices.

Recently, tissue slices have been adapted to study both mouse and human T cell development. Thymic slices combine and complement the strengths of existing organotypic culture systems to study thymocyte differentiation. Specifically, the thymic slice system allows for high throughput experiments and the ability to introduce homogenous developmental intermediate populations into an environment with a well-established cortex and medulla. These qualities make thymic slices a highly versatile and technically accessible model to study thymocyte development. Here we describe methods to prepare, embed, and slice thymic lobes to study T cell development in situ.

Opposing chemokine gradients control human thymocyte migration in situ.

The ordered migration of thymocytes from the cortex to the medulla is critical for the appropriate selection of the mature T cell repertoire. Most studies of thymocyte migration rely on mouse models, but we know relatively little about how human thymocytes find their appropriate anatomical niches within the thymus. Moreover, the signals that retain CD4+CD8+ double-positive (DP) thymocytes in the cortex and prevent them from entering the medulla prior to positive selection have not been identified in mice or humans. Here, we examined the intrathymic migration of human thymocytes in both mouse and human thymic stroma and found that human thymocyte subsets localized appropriately to the cortex on mouse thymic stroma and that MHC-dependent interactions between human thymocytes and mouse stroma could maintain the activation and motility of DP cells. We also showed that CXCR4 was required to retain human DP thymocytes in the cortex, whereas CCR7 promoted migration of mature human thymocytes to the medulla. Thus, 2 opposing chemokine gradients control the migration of thymocytes from the cortex to the medulla. These findings point to significant interspecies conservation in thymocyte-stroma interactions and provide the first evidence that chemokines not only attract mature thymocytes to the medulla, but also play an active role in retaining DP thymocytes in the cortex prior to positive selection.

T cells in cryptopatch aggregates share TCR gamma variable region junctional sequences with gamma delta T cells in the small intestinal epithelium of mice.

The role of cryptopatch aggregates in the development of intestinal intraepithelial lymphocytes (IEL) is a matter of controversy. Therefore, an important question is whether T cells in cryptopatch aggregates are lineally related to IEL. We hypothesized that if gammadelta+ IEL derive from T cells in cryptopatch aggregates, then a clonal relationship would exist between the two populations. To test this hypothesis, we compared the sequence of rearranged TCR gamma variable region 5 genes in gammadelta+ IEL and cryptopatch cells. We purified IEL by FACS and cryptopatch cells were isolated from frozen sections of the intestine by laser-assisted microdissection. PCR showed that TCR gamma variable region 5 was rearranged in gammadelta+ IEL and in CD3+ cryptopatch cells, but not in CD3- cryptopatch cells. DNA sequence analysis showed that the frequency of in-frame junctions in cryptopatch aggregates was at a level consistent with positive selection in both wild-type and athymic nude mice. In addition, the predicted amino acid sequences of V-J junctions present in gammadelta+ IEL and cryptopatch cells were encoded by identical nucleotide sequences. By contrast, the frequency of in-frame joints was significantly reduced in cryptopatch cells isolated from TCR delta-deficient mice, indicating that the enrichment of in-frame joints in cryptopatch cells must normally depend on expression of surface gammadelta TCR. Our results are consistent with the hypothesis that a subset of gammadelta+ IEL are related to T cells in cryptopatch aggregates. The precise role of cryptopatch aggregates in intestinal gammadelta+ T cell homeostasis still needs to be determined.