Jan C. Dudda

Dendritic Cell Immunization Route Determines CD8+ T Cell Trafficking to Inflamed Skin: Role for Tissue Microenvironment and Dendritic Cells in Establishment of T Cell-Homing Subsets

The effector/memory T cell pool branches in homing subsets selectively trafficking to organs such as gut or skin. Little is known about the critical factors in the generation of skin-homing CD8+ T cells, although they are crucial effectors in skin-restricted immune responses such as contact hypersensitivity and melanoma defense. In this study, we show that intracutaneous, but not i.v. injection of bone marrow-derived dendritic cells induced skin-homing CD8+ T cells with up-regulated E-selectin ligand expression and effector function in contact hypersensitivity. The skin-homing potential and E-selectin ligand expression remained stable in memory phase without further Ag contact. In contrast, i.p. injection induced T cells expressing the gut-homing integrin α4β7. Although differential expression of these adhesion molecules was strictly associated with the immunization route, the postulated skin-homing marker CCR4 was transiently up-regulated in all conditions. Interestingly, dendritic cells from different tissues effectively induced the corresponding homing markers on T cells in vitro. Our results suggest a crucial role for the tissue microenvironment and dendritic cells in the instruction of T cells for tissue-selective homing and demonstrate that Langerhans cells are specialized to target T cells to inflamed skin.

Toll-like receptor and IL-12 signaling control susceptibility to contact hypersensitivity

Allergic contact hypersensitivity (CHS) is a T cell–mediated inflammatory skin disease. Interleukin (IL)-12 is considered to be important in the generation of the allergen-specific T cell response. Loss of IL-12 function in IL-12Rβ2–deficient mice, however, did not ameliorate the allergic immune response, suggesting alternate IL-12–independent pathways in the induction of CHS. Because exposure to contact allergens always takes place in the presence of microbial skin flora, we investigated the potential role of Toll-like receptors (TLRs) in the induction of CHS. Using mice deficient in TLR4, the receptor for bacterial lipopolysaccharide (LPS), IL-12 receptor (R) β2, or both, we show that the concomitant absence of TLR4 and IL-12Rβ2, but not the absence of TLR4 or IL-12Rβ2 alone, prevented DC-mediated sensitization, generation of effector T cells, and the subsequent CHS response to 2,4,6-trinitro-1-chlorobenzene (TNCB), oxazolone, and fluorescein isothiocyanate. Introduction of the TLR4 transgene into the TLR4/IL-12Rβ2 mutant restored the CHS inducibility, showing a requirement for TLR4 in IL-12–independent CHS induction. Furthermore, the concomitant absence of TLR2 and TLR4 prevented the induction of CHS to TNCB in IL-12–competent mice. Finally, CHS was inducible in germ-free wild-type and IL-12Rβ2–deficient mice, but not in germ-free TLR4/IL-12Rβ2 double deficient mice, suggesting that the necessary TLR activation may proceed via endogenous ligands.

Dendritic cells govern induction and reprogramming of polarized tissue-selective homing receptor patterns of T cells: important roles for soluble factors and tissue microenvironments.

Tissue‐selective homing is established during naive T cell activation by the tissue microenvironment and tissue‐specific dendritic cells (DC). The factors driving induction and maintenance of T cell homing patterns are still largely unknown. Here we show that soluble factors produced during the interaction of T cells with CD11c+ DC isolated from skin‐ or small intestine‐associated tissues differentially modulate expression of the corresponding tissue‐selective homing receptors (E‐selectin ligands and α4β7 integrin/CCR9, respectively) on murine CD8+ T cells. Injection of tissue‐specific DC via different routes induces T cells with homing receptors characteristic of the corresponding local tissue microenvironment, independent of the origin of the DC. These data indicate an important role for signals delivered in trans. Moreover, DC can reprogram the homing receptor expression on T cells previously polarized in vitro for homing to skin or small intestine. Importantly, skin‐homing memory T cells stimulated directly ex vivo can also be reprogrammed by intestinal DC to a gut‐homing phenotype. Our results show that tissue‐selective homing receptor expression on effector and memory T cells is governed by inductive as well as suppressive signals from both DC and tissue microenvironments.

Foxp3+ regulatory T cells maintain immune homeostasis in the skin

Cutaneous immune responses must be tightly controlled to prevent unwanted inflammation in response to innocuous antigens, while maintaining the ability to combat skin-tropic pathogens. Foxp3+ regulatory T (T reg) cells are potent immune regulators and are found at high frequency in both human and mouse skin. Although T reg cells migrate to the skin and can dampen immune responses during experimentally induced inflammation or infection, the importance of cutaneous T reg cells for maintaining normal immune homeostasis in the skin has not been addressed. To selectively block T reg cell function in the skin, we restored the T reg cell compartment in Foxp3-deficient scurfy mice with cells whose ability to migrate to the skin was impaired because of targeted mutation of α-1,3-fucosyltransferase VII (Fut7). Although Fut7-deficient T reg cells were present at normal frequency and could function in all other tissues examined, these animals rapidly developed severe cutaneous inflammation. Thus, skin-resident T reg cell are essential for maintaining normal immune homeostasis at this site.

Induction of organ-selective CD4+ regulatory T cell homing

Compelling evidence suggests that Foxp3+CD25+CD4+ Treg play a fundamental role in immunoregulation. We have previously demonstrated that Treg have to enter peripheral tissues to suppress ongoing inflammation. However, relatively little is known about how Treg acquire the expression of homing receptors required for tissue‐ or inflammation‐specific migration. Migratory properties of conventional naïve T cells are shaped by the tissue microenvironment and organ‐specific dendritic cells during priming. Here, we show that this basic concept also holds true for CD25+CD4+ Treg: Priming of Treg within peripheral LN led to the expression of selectin ligands, which facilitate migration into inflamed skin, whereas activation within mesenteric LN led to induction of the integrin α4β7, which is required for migration into mucosal tissues. Furthermore, we could establish in vitro culture systems containing either dendritic cells from mesenteric and peripheral LN, or retinoic acid and IL‐12 as polarizing compounds to induce mucosa‐ and skin‐seeking Treg, respectively. Together, our results demonstrate that Treg, similarly to conventional T cells, can be configured with organ‐selective homing properties allowing efficient targeting into distinct tissues.

Cutting Edge: Instructive Role of Peripheral Tissue Cells in the Imprinting of T Cell Homing Receptor Patterns

Tissue-specific homing of effector and memory T cells to skin and small intestine requires the imprinting of specific combinations of adhesion molecules and chemokine receptors by dendritic cells in the draining lymph nodes. In this study, we demonstrate that CD8+ T cells activated by Ag-pulsed bone marrow-derived dendritic cells were induced to express the small intestine homing receptors α4β7 integrin and chemokine receptor CCR9 in coculture with small intestinal epithelial cells. In contrast, in coculture with dermal fibroblasts the skin-homing receptor E-selectin ligand was induced. Interestingly, the imprinting of gut homing receptors on anti-CD3/anti-CD28 stimulated T cells was induced by soluble factors produced by small intestinal epithelial cells. Retinoic acid was identified as a crucial factor. These findings show that peripheral tissue cells directly produce homing receptor imprinting factors and suggest that dendritic cells can acquire their imprinting potential already in the peripheral tissue of origin.

Administration of a Synthetic TLR4 Agonist Protects Mice from Pneumonic Tularemia

Francisella tularensis is a Gram-negative intracellular pathogen that causes the zoonosis tularemia. Because F. tularensis LPS causes weak TLR4 activation, we hypothesized that administration of a synthetic TLR4 agonist, aminoalkyl glucosaminide phosphate (AGP), would boost the innate immune system and compensate for reduced TLR4 stimulation. Intranasal administration of AGPs induced intrapulmonary production of proinflammatory cytokines and chemokines. Mice treated with AGPs before and after inhalation of Francisella novicida exhibited augmented cytokine and inflammatory responses to infection; reduced bacterial replication in lung, liver, and spleen; and increased survival, whereas all PBS-treated control mice died within 4 days of infection, all AGP-treated mice showed prolonged time-to-death, and 30–60% of AGP-treated mice survived. The protective effect of AGP was lost in mice lacking IFN-γ. Long-term survivors developed specific Th1 splenocyte responses and specific Abs dominated by IgG2 isotypes. Survivors were fully protected from rechallenge with aerosolized F. novicida. Thus, preventive administration of AGP successfully modulated innate immune responses to aerosolized F. novicida, leading to protective immunity to pneumonic tularemia. This is the first report of the protective effect of a TLR ligand on resistance to F. novicida-induced pneumonic tularemia.

Fas-Mediated Inhibition of CD4+ T Cell Priming Results in Dominance of Type 1 CD8+ T Cells in the Immune Response to the Contact Sensitizer Trinitrophenyl

One of the unusual properties of chemically reactive haptens is their capacity to simultaneously generate immunogenic determinants for hapten-specific CD8+ and CD4+ T cells. Surprisingly, however, a clear dominance of CD8+ effector T cells is observed in murine contact hypersensitivity to various haptens and upon T cell priming with hapten-modified APCs in vitro. In this study we show that trinitrophenyl-specific CD8+ T cells actively prevent CD4+ T cell priming in vitro. This process requires cell-cell contact and is dependent on the expression of Fas on the CD4+ T cells. Our results reveal an important Fas-dependent mechanism for the regulation of hapten-specific CD4+ T cell responses by CD8+ T cells, which causes the dominance of CD8+ effector T cells and the active suppression of a CD4+ T cell response. Moreover, our demonstration of reduced contact hypersensitivity to trinitrophenyl in the absence of Fas, but not of perforin and/or granzymes A and B, underlines the important role of Fas as a pathogenetic factor for contact hypersensitivity.

Early cytoskeletal rearrangement during dendritic cell maturation enhances synapse formation and Ca2+ signaling in CD8+ T cells

The interplay between dendritic cells (DC) and T cells is a dynamic process critically depending on DC maturation. Ca2+ influx is one of the initial events occurring during DC/T cell contacts. To determine how DC maturation influences DC/T cell contacts, time‐lapse video microscopy was established using TCR‐transgenic CD8+ T cells from P14 mice. DC maturation shifted DC/T cell contacts from short‐lived interactions with transient Ca2+ influx in T cells to long‐lasting interactions and sustained Ca2+ influx of 30 min and more. Follow‐up of DC/T cell interactions after 2 h using confocal microscopy revealed that long‐lasting Ca2+ responses in T cells were preferentially associated with the formation of an immunological synapse involving CD54 and H2‐Kb at the DC/T cell interface. Such synapse formation preceded MHC or B7 up‐regulation, since DC developed into potent Ca2+ stimulators 7 h after initiation of maturation. Instead, the enhanced capacity of 7 h‐matured DC to induce sustained Ca2+ responses in CD8+ T cells is critically dependent on the polarization and rearrangement of the cytoskeleton, as shown by Clostridium difficile toxin B inhibitor experiments. These data indicate that already very early after receiving a maturation stimulus, DC display enhanced cytoskeletal activity resulting in the rapid formation of immunological synapses and effective CD8+ T cell stimulation.

Efficiency of Dendritic Cell Vaccination against B16 Melanoma Depends on the Immunization Route

Dendritic cells (DC) presenting tumor antigens are crucial to induce potent T cell-mediated anti-tumor immune responses. Therefore DC-based cancer vaccines have been established for therapy, however clinical outcomes are often poor and need improvement. Using a mouse model of B16 melanoma, we found that the route of preventive DC vaccination critically determined tumor control. While repeated DC vaccination did not show an impact of the route of DC application on the prevention of tumor growth, a single DC vaccination revealed that both the imprinting of skin homing receptors and an enhanced proliferation state of effector T cells was seen only upon intracutaneous but not intravenous or intraperitoneal immunization. Tumor growth was prevented only by intracutaneous DC vaccination. Our results indicate that under suboptimal conditions the route of DC vaccination crucially determines the efficiency of tumor defense. DC-based strategies for immunotherapy of cancer should take into account the immunization route in order to optimize tissue targeting of tumor antigen specific T cells.