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Dendritic cells (DC) are critical for the initiation of immune responses; however, their role in priming IL-4–producing Th2 cells in vivo is not fully understood. We used a model of intradermal injection with fluorescent-labeled, nonviable larvae from the helminth parasite nonviable Nippostrongylus brasiliensis L3 larvae (Nb), a strong inducer of Th2 responses, together with IL-4–GFP reporter mice that enable a sensitive detection of IL-4 production to examine the contribution of DC to the priming of IL-4–producing CD4+ T cells in vivo. We found that parasite material is taken up by two distinct DC populations in draining lymph nodes: a mostly CD11cintMHC class II (MHCII)hiCD11b+Ly6C− dermal DC population and a CD11chiMHCIIintCD11b+Ly6C+ monocyte-derived DC population. After Nb treatment, these two DC populations appeared in the draining lymph nodes in comparable numbers and with similar kinetics; however, treatment with pertussis toxin blocked the migration of dermal DC and the priming of IL-4–producing T cells, but only partially affected monocyte-derived DC numbers. In line with this observation, transfer of OVA-loaded CD11cintMHCIIhi DC from Nb-treated mice into naive hosts could sensitize OVA-specific CD4+ T cells to IL-4 production, whereas transfer of CD11cintMHCIIhi DC from naive mice, or CD11chiMHCIIint DC from Nb-treated or naive mice, induced CD4+ T cell expansion but no IL-4 production. Phenotypic analysis of Nb-loaded CD11cintMHCIIhi DC revealed expression of programmed death ligand 2, CD301b, IFN regulatory factor 4, and moderate upregulation of OX40 ligand. However, thymic stromal lymphopoietin and OX40 ligand were not required for Th2 priming. Thus, our data suggest that appropriate stimuli can induce DC to express the unique signals sufficient to direct CD4+ T cells to Th2 differentiation.

Utilizing a sensitive and selective assay for IL-4, it was shown that lymph node T cells from naive mice could produce small amounts of this lymphokine in response to anti-CD3 antibodies adsorbed to culture dishes. The capacity of these cells to produce IL-4 in response to plate-bound anti-CD3 was substantially enhanced by the addition of IL-2 to the culture and was strikingly inhibited by monoclonal anti-IL-2 antibody. Thus, IL-2 appears to be essential for IL-4 production by anti-CD3 antibody-stimulated T cells from naive mice. The effect of IL-2 was not mediated either by preferential proliferation or survival of precursors of IL-4 producing cells, indicating that IL-2 regulates T cell production of IL-4. IL-4 producing capacity of T cells from naive mice was found mainly among CD4+ T cells. Large T cells produced much more IL-4, on a per cell basis, than did small T cells. In contrast, small T cells appeared to be equal or superior to large T cells in producing IL-2. The superiority of large T cells in IL-4-producing capacity was not accounted for by a lack of an accessory cell population from the small T cells as addition of large spleen cells depleted of both B and T cells did not enhance IL-4 production by small lymph node T cells. These results suggest that the bulk of IL-4 production by T cell populations, from normal mice, in response to anti-CD3 depends upon cells that are already activated and that IL-2 is required for such production.

Resistin-like molecules (RELM) belong to a family of secreted proteins that are expressed in multiple helminth infections with important effects on the host immune response. However the importance of human RELM proteins in helminth infections is less well understood. To investigate this, we utilized transgenic mice that expressed the human resistin gene (Tg+). Following infection with Nippostrongylus brasiliensis, a hookworm that colonizes the lung and intestine, Tg+ mice exhibited significant lung inflammation, characterized by increased inflammatory monocytes and elevated parasite burdens compared to control Tg- mice. Genome-wide transcriptional profiling of the infected lung tissue from Tg+ and Tg- mice revealed that genes associated with cytokine and TLR signaling were significantly elevated in Tg+ mice. To delineate the downstream cellular mediators of resistin, we performed a resistin binding and chemotaxis assay and identified monocytes as the main cell-types that were responsive to resistin. Moreover, monocytes sorted from the infected lung tissue of Tg+ mice expressed significantly higher TNFα compared to Tg- mice. In human studies, increased serum resistin was associated with higher helminth burdens and was positively correlated with inflammatory cytokines. Together, these studies identify a detrimental role for human resistin in instigating a non-protective inflammatory response that may be mediated by monocytes.

Dendritic cells are critical for the initiation of immune responses, however, their role in priming IL4-producing Th2 cells is controversial and remains an area of intense investigation. We found that after skin injection of the nematode parasite Nippostrongylus brasiliensis (Nb), a strong inducer of Th2 responses, Nb material is taken up by a population of dermal dendritic cells in lymph node, and that this population is necessary and sufficient for IL4 priming. Blocking the migration of dermal dendritic cells to the lymph node also prevented the priming of IL4-producing cells, and chicken ovalbumin (OVA)-loaded dDC from Nb-treated mice were sufficient to sensitize OVA-specific CD4+ T cells to IL4 production in vivo. Nb-loaded dermal dendritic cells were CD11b+, CD326neg, CD103neg, PDL2hi and IRF4+, and modestly upregulated expression of OX40L. However, OX40L and TSLP were not required for Th2 priming. Thus, our data suggest that the appropriate stimuli can induce DC to express the unique signals that direct CD4+ T cells to Th2 differentiation.

Type II immune responses are associated with protection against helminth infections, as well as the pathology of allergic diseases that are initiated against innocuous antigens. Two integral cytokines associated with type II immunity are interleukin (IL-)4 and IL-13. The 4C13R transgenic dual reporter mouse has been created to allow identification of IL-4 and IL-13 producing cells by the production of two intracellular fluorescent molecules, AmCyan and DS-Red respectively. The expression of the distinct reporter proteins are under the normal transcriptional control of the Il4 or Il13 genes. This technology allows for the analysis of in situ IL-4 and/or IL-13 production by the relevant differentiated immune cell types without any effect on the endogenous cytokine genes or their effector activities in the mouse. Using this reporter system we have identified that in vitro generated CD4+ Th2 cells have divergent expression of IL-4 and IL-13, suggesting cytokine specific Th2 subsets. Additionally, IL-13 expression is delayed compared with IL-4 in this system. Reporter expression in vivo has also highlighted that IL-4/IL-13 double producers are only a subpopulation of CD4+ T cells, and cytokine expression profiles differ significantly between lymph node and effector tissues during type II responses. Studying the expression and regulation of these cytokines will allow us to understand their contribution in both disease and protection.

Type 2 immunity is critical for defense against cutaneous infections, but also underlies the development of allergic skin diseases. We report the identification in normal murine dermis of an abundant, phenotypically unique group 2 innate lymphoid cell (ILC2) subset that depends on IL-7 and constitutively produces IL-13. Intravital multiphoton microscopy revealed that dermal ILC2 specifically interact with mast cells, whose function is suppressed by IL-13. Treatment of Rag1−/− mice with IL-2 led to a marked expansion of activated, IL-5-producing dermal ILC2, resulting in the development of spontaneous dermatitis characterized by eosinophil infiltrate and activated mast cells. Our data uncover a novel interactive pathway between two innate immune cell populations implicated in type 2 immunity, and demonstrate that ILC2 exhibit both pro- and anti-inflammatory properties.

We have examined the role of dendritic cells using different models of Th2 allergic immune responses in the skin. In the first model we used the nematode parasite Nippostrongylus brasiliensis, which infects rodent hosts by penetrating through the skin. Material from labelled N. brasiliensis is taken up by migratory dendritic cells and transported to the draining lymph node where it initiates a strong Th2 immune response. We have characterized the surface phenotype of these dendritic cells in terms of lineage markers, and for expression of markers associated with Th2 immune responses. We find that these dendritic cells express markers that are compatible with a steady-state population of dermal dendritic cells. In addition, we have carried out dendritic cell transfer experiments to show that these migratory dendritic cells maintain the full capacity to prime Th2 immune responses in vivo. Additional models of Th2 immune responses, such as those initiated by the powerful allergen House Dust Mite and by application of the vitamin D analogue MC903, are currently being investigated. Our data indicate that dendritic cells can carry a sufficient set of signals to instruct the initiation of Th2 immune responses in secondary lymphoid organs.

Basophils are recognized as immune modulators through their ability to produce IL-4, a key cytokine required for Th2 immunity. It has also recently been reported that basophils are transiently recruited into the draining LN after allergen immunization and that the recruited basophils promote the differentiation of naïve CD4 T cells into Th2 effector cells. Likewise, basophils were recruited into the draining mediastinal and mesenteric LN at 3 and 10 days after infection with intestinal nematode Nippostrongylus brasiliensis, respectively. However, the recruitment was completely abolished in mice deficient in IL-3 or IL-3Rβ, suggesting that the IL-3/IL-3R system is absolutely required. Activated CD4 T cells appeared to be the primary source of IL-3. Experiments using bone marrow chimera strategy further revealed that the cell types expressing IL-3Rβ are of bone marrow origin. Unexpectedly, the lack of IL-3 or of basophil LN recruitment played little role in N. brasiliensis-induced Th2 immune responses as unaltered Th2 immune responses were observed in N. brasiliensis infected IL-3-/- mice. Consistent with these findings, basophil depletion in infected mice did not diminish the development of IL-4-producing CD4 T cells. Therefore, our results reveal a previously unknown role of IL-3 in recruiting basophils to the LN and demonstrate that basophils are not necessarily associated with the development of Th2 immunity during parasite infection.

A mouse IL 3-dependent cell line, FD.C/1, can be induced to IL 2 growth responsiveness by culture in IL 2-conditioned culture medium. The IL 2-dependent cell lines derived by this procedure have been designated FD.C/2 cells. Once established, the FD.C/2 cells respond to human, rat, and mouse IL 2. When cultured with murine IL 3, FD.C/2 cells did not proliferate, appearing to accumulate in the G1 phase of the cell cycle. Other human and mouse lymphokines failed to stimulate FD.C/2 cell growth. The growth dependence of FD.C/2 cells on IL 2 could not be reversed to IL 3. Cell lines derived by these procedures could provide in vitro models for hemopoietic differentiative events.