Andrew G. Farr

A mechanism for the initiation of allergen-induced T helper type 2 responses

Both metazoan parasites and simple protein allergens induce T helper type 2 (TH2) immune responses, but the mechanisms by which the innate immune system senses these stimuli are unknown. In addition, the cellular source of cytokines that control TH2 differentiation in vivo has not been defined. Here we showed that basophils were activated and recruited to the draining lymph nodes specifically in response to TH2–inducing allergen challenge. Furthermore, we demonstrate that the basophil was the accessory cell type required for TH2 induction in response to protease allergens. Finally, we show that basophils were directly activated by protease allergens and produced TH2-inducing cytokines, including interleukin 4 and thymic stromal lymphopoietin, which are involved in TH2 differentiation in vivo.

T cell receptor δ gene mutant mice: Independent generation of αβ T cells and programmed rearrangements of γδ TCR genes

Abstract T cells bearing T cell receptor (TCR) γ and δ chain heterodimers are first generated early in ontogeny. They form distinct subsets that differ in their TCR repertoires and tissue distribution. Disruption of the mouse TCR Cδ gene segment by a gene targeting method caused the complete loss of T cells bearing TCR γδ chains, but had little or no effect on the development of T cells bearing TCR αβ chains. The analyses of TCR γ and δ genes in the mutant mice suggest that intracellular mechanisms acting at the level of DNA rearrangement play key roles in the differential γ and δ gene rearrangements and in the generation of the highly restricted junctional sequences during fetal thymic development.

Developmental regulation of Foxp3 expression during ontogeny

Thymectomy of neonatal mice can result in the development of autoimmune pathology. It has been proposed that thymic output of regulatory T (T reg) cells is delayed during ontogeny and that the development of autoimmune disease in neonatally thymectomized mice is caused by the escape of self-reactive T cells before thymectomy without accompanying T reg cells. However, the kinetics of T reg cell production within the thymus during ontogeny has not been assessed. We demonstrate that the development of Foxp3-expressing T reg cells is substantially delayed relative to nonregulatory thymocytes during ontogeny. Based on our data, we speculate that induction of Foxp3 in developing thymocytes and, thus, commitment to the T reg cell lineage is facilitated by a signal largely associated with the thymic medulla.

Cloning of a receptor subunit required for signaling by thymic stromal lymphopoietin

Signaling by type I cytokines involves the formation of receptor homodimers, heterodimers or higher order receptor oligomers. Here we report the cloning of a type I cytokine receptor subunit that is most closely related to the common cytokine receptor γ chain (γc). Binding and crosslinking experiments demonstrate that this protein is the receptor for a recently described interleukin 7 (IL-7)-like factor, thymic stromal lymphopoietin (TSLP). Binding of TSLP to the thymic stromal lymphopoietin receptor (TSLPR) is increased markedly in the presence of the IL-7 receptor α chain (IL-7Rα). IL-7Rα–expressing but not parental 32D cells proliferate in the presence of exogenous TSLP. Moreover, a combination of IL-7Rα and TSLPR is required for TSLP-dependent activation of a STAT5-dependent reporter construct. Thus it is shown that IL-7Rα is a component of both the IL-7 and TSLP receptors, which helps to explain why deletion of the gene that encodes IL-7Rα affects the lymphoid system more severely than deletion of the gene encoding IL-7 does. Cloning of TSLPR should facilitate an understanding of TSLP function and its signaling mechanism.

Lymph node-resident lymphatic endothelial cells mediate peripheral tolerance via Aire-independent direct antigen presentation.

Peripheral immune tolerance is generally thought to result from cross-presentation of tissue-derived proteins by quiescent tissue-resident dendritic cells to self-reactive T cells that have escaped thymic negative selection, leading to anergy or deletion. Recently, we and others have implicated the lymph node (LN) stroma in mediating CD8 T cell peripheral tolerance. We demonstrate that LN-resident lymphatic endothelial cells express multiple peripheral tissue antigens (PTAs) independent of the autoimmune regulator (Aire). They directly present an epitope derived from one of these, the melanocyte-specific protein tyrosinase, to tyrosinase-specific CD8 T cells, leading to their deletion. We also show that other LN stromal subpopulations express distinct PTAs by mechanisms that vary in their Aire dependence. These results establish lymphatic endothelial cells, and potentially other LN-resident cells, as systemic mediators of peripheral immune tolerance.

A role for pref-1 and HES-1 in thymocyte development.

T lymphocyte development requires a series of interactions between developing thymocytes and thymic epithelial (TE) cells. In this paper we show that TE cells in the developing thymus express Pref-1, a Delta-like cell-surface molecule. In fetal thymus organ cultures (FTOC), thymocyte cellularity was increased by the exogenous dimeric Pref-1 fusion protein, but was reduced by the soluble Pref-1 monomer or anti-Pref-1 Ab. Dimeric Pref-1 in FTOC also increased thymocyte expression of the HES-1 transcription factor. Thymocyte cellularity was increased in FTOC repopulated with immature thymocytes overexpressing HES-1, whereas FTOC from HES-1-deficient mice were hypocellular and unresponsive to the Pref-1 dimer. We detected no effects of either Pref-1 or HES-1 on developmental choice among thymocyte lineages. These results indicate that Pref-1 expressed by TE cells and HES-1 expressed by thymocytes are critically involved in supporting thymocyte cellularity.

Differentiation of regulatory Foxp3+ T cells in the thymic cortex.

Regulatory Foxp3+ T cells (TR) are indispensable for preventing autoimmune pathology in multiple organs and tissues. During thymic differentiation T cell receptor (TCR)–ligand interactions within a certain increased affinity range, in conjunction with γc-containing cytokine receptor signals, induce Foxp3 expression and thereby commit developing thymocytes to the TR lineage. The contribution of distinct MHC class II–expressing accessory cell types to the differentiation process of Foxp3+ thymocytes remains controversial, because a unique role in this process has been ascribed to either thymic dendritic cells (tDC) or to medullary thymic epithelial cells (mTEC). Furthermore, it was suggested that the thymic medulla, where the bulk of the negative selection of self-reactive thymocytes takes place, provides a specialized microenvironment supporting TR differentiation. Here, we report that the cortex, as defined by cortical thymic epithelial cells (cTEC), is sufficient for supporting TR differentiation. MHC class II expression restricted to both cTEC and mTEC or to cTEC alone did not significantly affect the numbers of Foxp3+ thymocytes. Furthermore, genetic or pharmacologic blockade of thymocyte migration resulted in a prominent accumulation of Foxp3+ thymocytes in the cortex, demonstrating that secondary signals required for Foxp3 up-regulation exist in the cortex. Our results suggest that mTEC or tDC do not serve as a cell type singularly responsible for TR differentiation and that neither the cortex nor the medulla exclusively provides an environment suitable for Foxp3 induction. Instead, multiple accessory cell types probably contribute to the thymic generation of regulatory Foxp3+ T cells.

Notch-Deficient Skin Induces a Lethal Systemic B-Lymphoproliferative Disorder by Secreting TSLP, a Sentinel for Epidermal Integrity

Epidermal keratinocytes form a highly organized stratified epithelium and sustain a competent barrier function together with dermal and hematopoietic cells. The Notch signaling pathway is a critical regulator of epidermal integrity. Here, we show that keratinocyte-specific deletion of total Notch signaling triggered a severe systemic B-lymphoproliferative disorder, causing death. RBP-j is the DNA binding partner of Notch, but both RBP-j–dependent and independent Notch signaling were necessary for proper epidermal differentiation and lipid deposition. Loss of both pathways caused a persistent defect in skin differentiation/barrier formation. In response, high levels of thymic stromal lymphopoietin (TSLP) were released into systemic circulation by Notch-deficient keratinocytes that failed to differentiate, starting in utero. Exposure to high TSLP levels during neonatal hematopoiesis resulted in drastic expansion of peripheral pre- and immature B-lymphocytes, causing B-lymphoproliferative disorder associated with major organ infiltration and subsequent death, a previously unappreciated systemic effect of TSLP. These observations demonstrate that local skin perturbations can drive a lethal systemic disease and have important implications for a wide range of humoral and autoimmune diseases with skin manifestations.

Thymic function in young/old chimeras: substantial thymic T cell regenerative capacity despite irreversible age-associated thymic involution

Age‐associated thymic involution results in a diminished capacity to regenerate T cell populations, although the magnitude of this effect is unknown. In this report, thymic function was studied in aged vs.young adult mice after lethal irradiation and administration of T cell‐depleted bone marrow (BM) from young mice. Abnormalities observed in aged thymi (reduced thymocyte numbers, histologic abnormalities) were not reversed by administration of young BM via bone marrow transplantation (BMT), but agend thymi displayed a normal thymocyte subset distribution and appropriately deleted Mls‐reactive T cells after BMT. Aged BMT recipients regenerated significantly reduced numbers of splenic T cells compared to young recipients and showed increased peripheral expansion of thymic emigrants since a higher proportion of BM‐derived T cells expressed a memory phenotype in aged vs.young BMT recipients. Because peripheral expansion of thymic emigrants could substantially increase the number of thymic progeny present in the spleen, we sought to measure thymic T cell regenerative capacity after BMT in a setting devoid of peripheral expansion. To do this, TCR‐transgenic (Tg+) T cell‐depleted BM was administered to aged and young recipients lacking antigen specific for the Tg+ TCR. Aged recipients regenerated approximately 50  % of the TCR Tg+ cells regenerated in young BMT recipients, providing evidence that even very aged thymi retain the capacity to regenerate significant numbers of mature T cell progeny. Therefore, thymic function is reduced with aged but it is not lost, suggesting that therapeutic approaches to enhance thymic function may be successful even in very aged hosts.

Thymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice.

Inherited deficiency of the CD40 ligand (X-linked hyper-IgM syndrome) is characterized by failure of immunoglobulin isotype switching and severe defects of cell-mediated immunity. To test the potential for gene transfer therapy to correct this disorder, we transduced murine bone marrow or thymic cells with a retroviral vector containing the cDNA for the murine CD40 ligand (CD40L) and injected them into CD40L–/– mice. Even low-level, constitutive expression of the transgene stimulated humoral and cellular immune functions in these mice. With extended follow-up, however, 12 of 19 treated mice developed T-lymphoproliferative disorders, ranging from polyclonal increases of lymphoblasts to overt monoclonal T-Lymphoblastic lymphomalymphomas that involved multiple organs. Our findings show that constitutive (rather than tightly regulated), low-level expression of CD40L can produce abnormal proliferative responses in developing T lymphocytes, apparently through aberrant interaction between CD40L+ and TCRαβ+CD40+ thymocytes. Current methods of gene therapy may prove inappropriate for disorders involving highly regulated genes in essential positions in proliferative cascades.