Immunoregulation in the tissues by gammadelta T cells.

For a T-cell subset to be classified as immunoregulatory, it might reasonably be predicted that in its absence, animals would experience pathological immune dysregulation. Moreover, reconstitution of the subset should restore normal immune regulation. So far, these criteria have been satisfied by only a few of the candidate regulatory T-cell subsets, but among them is the intraepithelial gammadelta T-cell receptor (TCR)+ subset of mouse skin. In this article, we look at immunoregulatory gammadelta T cells, and the growing evidence for tissue-associated immunoregulation mediated by both gammadelta T cells and alphabeta T cells.

Activation of GILZ gene by photoactivated 8-methoxypsoralen: potential role of immunoregulatory dendritic cells in extracorporeal photochemotherapy.

Extracorporeal photochemotherapy (ECP) is a widely used method for either immunization against cutaneous T cell lymphoma or immunosuppression of graft-versus-host disease and organ transplant rejection (OTR). Leukapheresed blood is routed through a chamber, in which 8-methoxypsoralen is activated by ultraviolet energy (PUVA), thereby causing DNA crosslinks in processed leukocytes. Return of ECP-processed mononuclear leukocytes to the patient then modulates aberrant T cell immunity. Since interaction with the ECP flow chamber induces monocyte-to-dendritic antigen presenting cell (DC) maturation, we examined the possibility that PUVA may direct the most heavily exposed monocytes to differentiate into tolerogenic DC, while the least exposed DC might remain immunogenic. Expression of the glucocorticoid-induced leucine zipper (GILZ) gene is a distinguishing marker of tolerogenic DC. We report that PUVA directly stimulates GILZ expression. PUVA-exposed DC up-regulated GILZ, down-regulated costimulatory CD80 and CD86, became resistant to Toll-like receptor-induced maturation, increased IL-10 production and decreased IL-12p70 production, all features of immunosuppressive DC. Knockdown of GILZ with siRNA reduced IL-10 and increased IL-12p70 production, demonstrating that GILZ is critical for this profile. PUVA-induction of GILZ expression by DC may help explain how ECP suppresses GVHD and OTR. Conversely, those ECP-processed monocytes minimally exposed to PUVA may mediate ECP's immunogenic effects.

Induction of monocyte-to-dendritic cell maturation by extracorporeal photochemotherapy: initiation via direct platelet signaling.

Extracorporeal Photochemotherapy (ECP) is a widely used therapy for cutaneous T cell lymphoma (CTCL). Although the mechanism of clinical action of ECP is not precisely established, previous studies have shown evidence of induction of dendritic cells (DCs). Here we show that, under flow conditions similar to those in post-capillary venules, ECP promotes platelet immobilization and activation, initiating stepwise receptor-ligand interactions with monocytes, which then differentiate into DC. These findings clarify how ECP directly stimulates DC maturation; suggest a new clinically applicable approach to the obtainment of DC; and identify a novel mechanism that may reflect physiological induction of DC.

Skint-1 is a highly specific, unique selecting component for epidermal T cells.

αß T-cell repertoire selection is mediated by peptide-MHC complexes presented by thymic epithelial or myeloid cells, and by lipid-CD1 complexes expressed by thymocytes. γδ T-cell repertoire selection, by contrast, is largely unresolved. Mice mutant for Skint-1, a unique Ig superfamily gene, do not develop canonical Vγ5Vδ1(+) dendritic epidermal T cells. This study shows that transgenic Skint-1, across a broad range of expression levels, precisely and selectively determines the Vγ5Vδ1(+) dendritic epidermal T-cell compartment. Skint-1 is expressed by medullary thymic epithelial cells, and unlike lipid-CD1 complexes, must be expressed by stromal cells to function efficiently. Its unusual transmembrane-cytoplasmic regions severely limit cell surface expression, yet increasing this or, conversely, retaining Skint1 intracellularly markedly compromises function. Each Skint1 domain appears nonredundant, including a unique decamer specifying IgV-domain processing. This investigation of Skint-1 biology points to complex events underpinning the positive selection of an intraepithelial γδ repertoire.

Skin immune surveillance by T cells--a new order?

Although studies of the skin have provided fundamental models for innate and adaptive immune surveillance of body surfaces, there remains relatively little understanding of the role that epithelial cells play in sensing infection and/or organ dysfunction, and the pathways available to them to communicate with local and systemic immune cells. In particular, evidence is emerging for a novel stress response initiated by local lymphocytes, rather than dendritic cells, and based on their recognition of epithelial stress-induced antigens. Its consequences are to sustain tissue integrity by providing immunoprotection and novel modes of immunoregulation, whereas its dysregulation may promote body surface immunopathologies.

Rapid generation of maturationally synchronized human dendritic cells: contribution to the clinical efficacy of extracorporeal photochemotherapy.

Extracorporeal photochemotherapy (ECP) is widely used to treat cutaneous T-cell lymphoma, graft-versus-host disease, and allografted organ rejection. Its clinical and experimental efficacy in cancer immunotherapy and autoreactive disorders suggests a novel mechanism. This study reveals that ECP induces a high percentage of processed monocytes to enter the antigen-presenting dendritic cell (DC) differentiation pathway, within a single day, without added cytokines, as determined by enhanced expression of relevant genes. The resulting DCs are capable of processing and presentation of exogenous and endogenous antigen and are largely maturationally synchronized, as assessed by the level of expression of costimulatory surface molecules. Principal component analysis of the ECP-induced monocyte transcriptome reveals that activation or suppression of more than 1100 genes produces a reproducible distinctive molecular signature, common to ECP-processed monocytes from normal subjects, and those from patients. Because ECP induces normal monocytes to enter the DC differentiation pathway, this phenomenon is independent of disease state. The efficiency with which ECP stimulates new functional DCs supports the possibility that these cells participate prominently in the clinical successes of the treatment. Appropriately modified by future advances, ECP may potentially offer a general source of therapeutic DCs.

Role of macrophage migration inhibitory factor in the Th2 immune response to epicutaneous sensitization.

We examined the role of macrophage migration inhibitory factor (MIF) in the generation of the Th2 response using MIF-deficient mice in a model of epicutaneous sensitization to ovalbumin. Lymph node cells from sensitized MIF-deficient mice produce lower levels of Th2 cytokines after antigen challenge when compared to their wild-type counterparts. Sensitized mice lacking MIF show less pulmonary inflammation after intranasal antigen exposure. Mice deficient in CD74, the MIF receptor, also are unable to generate an inflammatory response to epicutaneous sensitization. Examination of the elicitation phase of the atopic response using DO11.10 OVA TCR transgenic animals shows that T cell proliferation and IL-2 production are strongly impaired in MIF-deficient T cells. This defect is most profound when both T cells and antigen-presenting cells are lacking MIF. These data suggest that MIF is crucial both for the sensitization and the elicitation phases of a Th2-type immune response in allergic disease.

Age-dependent requirement for gammadelta T cells in the primary but not secondary protective immune response against an intestinal parasite.

Between weaning (3 wk of age) and adulthood (7 wk of age), mice develop increased resistance to infection with Eimeria vermiformis, an abundant intestinal parasite that causes coccidiosis. This development of resistance was perturbed in T cell receptor (TCR)delta(-/-) mice, which at 4 wk of age remained largely susceptible to infection and prone to infection-associated dehydration. These phenotypes were rescued by the repopulation of gammadelta cells after adoptive transfer of lymphoid progenitors into newborn recipients. Because alphabeta T cells are necessary and sufficient for the protection of adult mice against E. vermiformis, the requirement for gammadelta cells in young mice shows a qualitative difference between the cellular immune responses operating at different ages. An important contribution toward primary immune protection in young hosts may have provided a strong selective pressure for the evolutionary conservation of gammadelta cells. This notwithstanding, the development of effective, pathogen-specific immunity in young mice requires alphabeta T cells, just as it does in adult mice.

The distinct contributions of murine T cell receptor (TCR)gammadelta+ and TCRalphabeta+ T cells to different stages of chemically induced skin cancer.

Epithelial tissues in which carcinomas develop often contain systemically derived T cell receptor (TCR)alphabeta+ cells and resident intraepithelial lymphocytes that are commonly enriched in TCRgammadelta+ cells. Recent studies have demonstrated that gammadelta cells protect the host against chemically induced cutaneous malignancy, but the role of alphabeta T cells has been enigmatic, with both protective and tumor-enhancing contributions being reported in different systems. This study aims to clarify the contributions of each T cell type to the regulation of squamous cell carcinoma induced in FVB mice by a two-stage regimen of 7,12-dimethylbenz[a]anthracene initiation followed by repetitive application of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate. This protocol permits one to monitor the induction of papillomas and the progression of those papillomas to carcinomas. The results show that whereas gammadelta cells are strongly protective, the nonredundant contributions of alphabeta T cells to the host's protection against papillomas are more modest. Furthermore, at both high and low doses of carcinogens, alphabeta T cells can contribute to rather than inhibit the progression of papillomas to carcinomas. As is likely to be the case in humans, this study also shows that the contribution of T cells to tumor immunosurveillance is regulated by modifier genes.

Resident skin-specific gammadelta T cells provide local, nonredundant regulation of cutaneous inflammation.

The function of the intraepithelial lymphocyte (IEL) network of T cell receptor (TCR) gammadelta(+) (Vgamma5(+)) dendritic epidermal T cells (DETC) was evaluated by examining several mouse strains genetically deficient in gammadelta T cells (delta(-/-) mice), and in delta(-/-) mice reconstituted with DETC or with different gammadelta cell subpopulations. NOD.delta(-/-) and FVB.delta(-/-) mice spontaneously developed localized, chronic dermatitis, whereas interestingly, the commonly used C57BL/6.delta(-/-) strain did not. Genetic analyses indicated a single autosomal recessive gene controlled the dermatitis susceptibility of NOD.delta(-/-) mice. Furthermore, allergic and irritant contact dermatitis reactions were exaggerated in FVB.delta(-/-), but not in C57BL/6.delta(-/-) mice. Neither spontaneous nor augmented irritant dermatitis was observed in FVB.beta(-/-) delta(-/-) mice lacking all T cells, indicating that alphabeta T cell-mediated inflammation is the target for gammadelta-mediated down-regulation. Reconstitution studies demonstrated that both spontaneous and augmented irritant dermatitis in FVB.delta(-/-) mice were down-regulated by Vgamma5(+) DETC, but not by epidermal T cells expressing other gammadelta TCRs. This study demonstrates that functional impairment at an epithelial interface can be specifically attributed to absence of the local TCR-gammadelta(+) IEL subset and suggests that systemic inflammatory reactions may more generally be subject to substantial regulation by local IELs.

Development of immunogenic tumor-loaded dendritic cells through physical perturbation and apoptotic cell loading.

To improve understanding of the forces that drive monocytes to transition into dendritic cells (Liyanage et al., 2002), we developed an experimental system that converts monocytes to DC by passage of leukocytes through a 400 microm silica bead column. The results demonstrate that overnight culture of column-treated monocytes causes a phenotypic conversion that is characteristically displayed by immature DC. These phenotypic changes were enhanced when the DC were loaded with apoptotic cells, leading to increased expression of the DC maturation-associated markers CD83, CD80 and the chemokine receptor CCR7. The DC demonstrated potent induction of allogeneic T cell proliferation and the capacity to activate autologous CD8(+) T cells. The CD8 T cells expressed augmented levels of perforin, IFN-gamma and TNF-alpha and mediated CTCL cell apoptosis. These studies demonstrate that physical contact with silica beads combined with loading of apoptotic tumor cells induces synchronized, rapid conversion of human monocytes to DC, which can efficiently stimulate CD8(+) T cells. These results may aid in the development of more efficient DC vaccines that can be loaded with the universe of antigens available in apoptotic tumor cells in a rapid, clinically practical fashion.

The integration of conventional and unconventional T cells that characterizes cell-mediated responses.

This review builds on evidence that cell-mediated immune responses to bacteria, viruses, parasites, and tumors are an integration of conventional and unconventional T-cell activities. Whereas conventional T cells provide clonal antigen-specific responses, unconventional T cells profoundly regulate conventional T cells, often suppressing their activities such that immunopathology is limited. By extrapolation, immunopathologies and inflammatory diseases may reflect defects in regulation by unconventional T cells. To explore the function of unconventional T cells, several extensive gene expression analyses have been undertaken. These studies are reviewed in some detail, with emphasis on the mechanisms by which unconventional T cells may exert their regulatory functions. Highlighting the fundamental nature of T-cell integration, we also review emerging data that the development of conventional and unconventional T cells is also highly integrated.

Environmentally responsive and reversible regulation of epidermal barrier function by gammadelta T cells.

The intraepithelial lymphocyte (IEL) network possibly composes the largest T-cell compartment in the body, but it is poorly understood. IELs show limited T-cell receptor (TCR) diversity and have been proposed to respond to generic stress signals rather than pathogen-specific antigens. Consistent with this, skin-resident TCRgammadelta+ cells, known as dendritic epidermal T cells (DETC), downregulate cutaneous inflammation, promote wound healing, and protect against cutaneous neoplasia. These pleiotropic effects collectively suggest that DETC (and IEL more generally) may contribute to epithelial maintenance and barrier function. The present studies test this hypothesis. Using skin surface impedance analysis to measure hydration status and transepidermal water loss, we show that the epidermal barrier is defective in gammadelta T-cell deficient mice. However, this does not represent a constitutive role of gammadelta cells, but rather one that is dependent on environmental challenge, consistent with the primary role for lymphocytes being the response of the host to its environment. Likewise, the importance of the physiologic DETC-associated TCR is demonstrated by showing that Vgamma5+ fetal thymocytes reconstitute the barrier function defect in TCRdelta-/- mice, while Vgamma5-/- mice also show environmentally responsive defects in cutaneous physiology.

Generation of dendritic cells from rabbit bone marrow mononuclear cell cultures supplemented with hGM-CSF and hIL-4.

The in vitro generation of dendritic cells (DCs) from either blood or bone marrow has been accomplished for humans and a number of other species. This ability has facilitated the opportunity to test the efficacy of DC vaccines in various tumor models. The cottontail rabbit papillomavirus (CRPV) model is the most clinically relevant animal model for human papillomavirus (HPV)-associated carcinogenesis. The CRPV model has been used to test various preventative and therapeutic vaccination strategies, and the availability of rabbit DCs would further expand its utility. However, to date, rabbit DCs have not been phenotypically and/or functionally characterized. Here we show that DCs can be generated in vitro from rabbit bone marrow mononuclear cells (BMMCs) cultured in the presence of the human cytokines GM-CSF and IL-4 and matured with lipopolysaccharide (LPS). These cells show upregulation of MHC class II and CD86, as well as downregulation of CD14, do not have non-specific esterase activity, are able to perform receptor-mediated endocytosis, and are potent stimulators of allogeneic T cell proliferation in mixed lymphocyte reactions. The ability to generate rabbit DCs makes it possible to test the efficacy of DC vaccination in the prevention and treatment of CRPV-induced lesions, which may provide useful preclinical data regarding the use of DC vaccines for HPV-associated lesions, including cervical cancer.

Subset-specific, uniform activation among V gamma 6/V delta 1+ gamma delta T cells elicited by inflammation.

The V gamma 6/V delta 1(+) cells, the second murine gamma delta T cell subset to arise in the thymus, express a nearly invariant T cell receptor (TCR), colonize select tissues, and expand preferentially in other tissues during inflammation. These cells are thought to help in regulating the inflammatory response. Until now, V gamma 6/V delta 1(+) cells have only been detectable indirectly, by expression of V gamma 6-encoding mRNA. Here, we report that 17D1, a monoclonal antibody, which detects the related epidermis-associated V gamma 5/V delta 1(+) TCR, will also bind the V gamma 6/V delta 1(+) cells if their TCR is first complexed to an anti-C delta antibody. Features of this special condition for recognition suggest the possibility that an alternate structure exists for the V gamma 6/V delta 1 TCR, which is stabilized upon binding to the anti-C delta antibody. Using the 17D1 antibody as means to track this gamma delta T cell subset by flow cytometry, we discovered that the response of V gamma 6/V delta 1(+) cells during inflammation often far exceeds that of other subsets and that the responding V gamma 6/V delta 1(+) cells display a strikingly uniform activation/memory phenotype compared with other gamma delta T cell subsets.

Mechanisms of chemical cooperative carcinogenesis by epidermal Langerhans cells.

Cutaneous squamous cell carcinoma (SCC) is the most prevalent invasive malignancy with metastatic potential. The epidermis is exposed to a variety of environmental DNA-damaging chemicals, principal among which are polyaromatic hydrocarbons (PAHs) ubiquitous in the environment, tobacco smoke, and broiled meats. Langerhans cells (LCs) comprise a network of dendritic cells situated adjacent to basal, suprabasal, and follicular infundibular keratinocytes that when mutated can give rise to SCC, and LC-intact mice are markedly more susceptible than LC-deficient mice to chemical carcinogenesis provoked by initiation with the model PAH, 7,12-dimethylbenz[a]anthracene (DMBA). LCs rapidly internalize and accumulate DMBA as numerous membrane-independent cytoplasmic foci. Repopulation of LC-deficient mice using fetal liver LC-precursors restores DMBA-induced tumor susceptibility. LC expression of p450 enzyme CYP1B1 is required for maximal rapid induction of DNA-damage within adjacent keratinocytes and their efficient neoplastic transformation; however, effects of tumor progression also attributable to the presence of LC were revealed as CYP1B1 independent. Thus, LCs make multifaceted contributions to cutaneous carcinogenesis, including via the handling and metabolism of chemical mutagens. Such findings suggest a cooperative carcinogenesis role for myeloid-derived cells resident within cancer susceptible epithelial tissues principally by influencing early events in malignant transformation.

Characterizing the protective component of the alphabeta T cell response to transplantable squamous cell carcinoma.

There is increasing promise that cellular immune response may be manipulated to combat cancer; however, it is also clear that the immune response to cutaneous malignancy comprises different T cell activities that variably inhibit or promote tumor development. Thus, a better understanding of each of these activities is crucial to more effective clinical manipulation. To better characterize the protective anti-tumor effects of alphabeta T cells, we examined the growth of the transplantable squamous cell carcinoma (SCC) line, PDV, which is markedly inhibited in immunocompetent versusalphabeta T cell-deficient mice. We show that the protective response is composed of CD8(+) and interferon-gamma (IFNgamma)-producing CD4(+) cells, and that the most overt effects of these components on tumor growth in situ are to provoke overt focal necroses and to decrease the stromal bed. Tumors growing in the presence of any of these components also show reduced expression of Rae-1, a ligand for the activating NK receptor, NKG2D. Collectively, these data illustrate which components of the alphabeta T cell response against SCC have protective potential, and indicate which aspects of tumor physiology may be most susceptible to their activities.