Integrin-driven monocyte to dendritic cell conversion in modified extracorporeal photochemotherapy.

Due to clinical efficacy and safety profile, extracorporeal photochemotherapy (ECP) is a commonly used cell treatment for patients with cutaneous T cell lymphoma (CTCL) and graft-versus-host disease (GVHD). The capacity of ECP to induce dendritic antigen-presenting cell (DC)-mediated selective immunization or immunosuppression suggests a novel mechanism involving pivotal cell signalling processes that have yet to be clearly identified as related to this procedure. In this study we employ two model systems of ECP to dissect the role of integrin signalling and adsorbed plasma proteins in monocyte-to-DC differentiation. We demonstrate that monocytes that were passed through protein-modified ECP plates adhered transiently to plasma proteins, including fibronectin, adsorbed to the plastic ECP plate and activated signalling pathways that initiate monocyte-to-DC conversion. Plasma protein adsorption facilitated 54·2 ± 4·7% differentiation, while fibronectin supported 29·8 ± 7·2% differentiation, as detected by DC phenotypic expression of membrane CD80 and CD86, as well as CD36, human leucocyte antigen D-related (HLA-DR) and cytoplasmic CD83. Further, we demonstrate the ability of fibronectin and other plasma proteins to act through cell adhesion via the ubiquitous arginine-glycine-aspartic (RGD) motif to drive monocyte-to-DC differentiation, with high-density RGD substrates supporting 54·1 ± 5·8% differentiation via αVß3 and α5ß1integrin signalling. Our results demonstrate that plasma protein binding integrins and plasma proteins operate through specific binding domains to induce monocyte-to-DC differentiation in ECP, providing a mechanism that can be harnessed to enhance ECP efficacy.

Regulation of cutaneous malignancy by gammadelta T cells.

The localization of gammadelta T cells within epithelia suggests that these cells may contribute to the down-regulation of epithelial malignancies. We report that mice lacking gammadelta cells are highly susceptible to multiple regimens of cutaneous carcinogenesis. After exposure to carcinogens, skin cells expressed Rae-1 and H60, major histocompatibility complex-related molecules structurally resembling human MICA. Each of these is a ligand for NKG2d, a receptor expressed by cytolytic T cells and natural killer (NK) cells. In vitro, skin-associated NKG2d+ gammadelta cells killed skin carcinoma cells by a mechanism that was sensitive to blocking NKG2d engagement. Thus, local T cells may use evolutionarily conserved proteins to negatively regulate malignancy.

Development of dendritic epidermal T cells with a skewed diversity of gamma delta TCRs in V delta 1-deficient mice.

One of the most intriguing features of gammadelta T cells that reside in murine epithelia is the association of a specific Vgamma/Vdelta usage with each epithelial tissue. Dendritic epidermal T cells (DETCs) in the murine epidermis, are predominantly derived from the "first wave" Vgamma5+ fetal thymocytes and overwhelmingly express the canonical Vgamma5/Vdelta1-TCRs lacking junctional diversity. Targeted disruption of the Vdelta1 gene resulted in a markedly impaired development of Vgamma5+ fetal thymocytes as precursors of DETCs; however, gammadeltaTCR+ DETCs with a typical dendritic morphology were observed in Vdelta1-/- mice and their cell densities in the epidermis were slightly lower than those in Vdelta1+/- epidermis. Moreover, the Vdelta1-deficient DETCs were functionally competent in their ability to up-regulate cytokines and keratinocyte growth factor-expression in response to keratinocytes. Vgamma5+ DETCs were predominant in the Vdelta1-/- epidermis, though Vgamma5- gammadeltaTCR+ DETCs were also detected. The Vgamma5+ DETCs showed a typical dendritic shape, gammadeltaTCR(high), and age-associated expansion in epidermis as observed in conventional DETCs of normal mice, whereas the Vgamma5- gammadeltaTCR+ DETCs showed a less dendritic shape, gammadeltaTCR(low), and no expansion in the epidermis, consistent with their immaturity. These results suggest that optimal DETC development does not require a particular Vgamma/Vdelta-chain usage but requires expression of a limited diversity of gammadeltaTCRs, which allow DETC precursors to mature and expand within the epidermal microenvironment.

Granulocyte-macrophage colony-stimulating factor priming plus papillomavirus E6 DNA vaccination: effects on papilloma formation and regression in the cottontail rabbit papillomavirus--rabbit model.

A cottontail rabbit papillomavirus (CRPV) E6 DNA vaccine that induces significant protection against CRPV challenge was used in a superior vaccination regimen in which the cutaneous sites of vaccination were primed with an expression vector encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that induces differentiation and local recruitment of professional antigen-presenting cells. This treatment induced a massive influx of major histocompatibility complex class II-positive cells. In a vaccination-challenge experiment, rabbit groups were treated by E6 DNA vaccination, GM-CSF DNA inoculation, or a combination of both treatments. After two immunizations, rabbits were challenged with CRPV at low, moderate, and high stringencies and monitored for papilloma formation. As expected, all clinical outcomes were monotonically related to the stringency of the viral challenge. The results demonstrate that GM-CSF priming greatly augmented the effects of CRPV E6 vaccination. First, challenge sites in control rabbits (at the moderate challenge stringency) had a 0% probability of remaining disease free, versus a 50% probability in E6-vaccinated rabbits, and whereas GM-CSF alone had no effect, the interaction between GM-CSF priming and E6 vaccination increased disease-free survival to 67%. Second, the incubation period before papilloma onset was lengthened by E6 DNA vaccination alone or to some extent by GM-CSF DNA inoculation alone, and the combination of treatments induced additive effects. Third, the rate of papilloma growth was reduced by E6 vaccination and, to a lesser extent, by GM-CSF treatment. In addition, the interaction between the E6 and GM-CSF treatments was synergistic and yielded more than a 99% reduction in papilloma volume. Finally, regression occurred among the papillomas that formed in rabbits treated with the E6 vaccine and/or with GM-CSF, with the highest regression frequency occurring in rabbits that received the combination treatment.

Enterocyte expression of interleukin 7 induces development of gammadelta T cells and Peyer's patches.

The intestinal mucosa is suggested to support extrathymic T cell development, particularly for T cell receptor (TCR)-gammadelta intraepithelial lymphocytes (IELs). TCR-gammadelta cell development requires interleukin (IL)-7; IL-7(-/)- or IL-7 receptor(-/)- mice lack TCR-gammadelta cells. Using the intestinal fatty acid binding protein (iFABP) promoter, we reinstated expression of IL-7 to mature enterocytes of IL-7(-/)- mice (iFABP-IL7). In iFABP-IL7 mice, TCR-gammadelta IELs were restored, as were cryptopatches and Peyer's patches. TCR-gammadelta cells remained absent from all other tissues. Likewise, T cell development in thymus and B cell maturation in the bone marrow and spleen retained the IL-7(-/)- phenotype. Thus, IL-7 expression by enterocytes was sufficient for extrathymic development of TCR-gammadelta cells in situ within the intestinal epithelium and was crucial for organization of mucosal lymphoid tissue.

Th2 responses induced by epicutaneous or inhalational protein exposure are differentially dependent on IL-4.

Atopic individuals are predisposed to mounting vigorous Th2-type immune responses to environmental allergens. To determine the factors responsible, animal models that closely mimic natural modes of allergen exposure should prove most informative. Therefore, we investigated the role of IL-4, a known Th2-promoting cytokine, in generation of Th2 responses after exposure of either the skin or airway to soluble protein. Compared with wild-type (WT) mice, IL-4-deficient (IL-4(-/-)) mice showed markedly impaired Th2 activation after primary exposure to inhaled ovalbumin (OVA), with decreased OVA-specific IgG1 and IgE, and significantly fewer eosinophils in bronchoalveolar lavage (BAL) fluid after airway challenge. In contrast, IL-4(-/-) mice initially exposed to epicutaneous (e.c.) OVA mounted Th2 responses equivalent to responses in WT mice, with high numbers of eosinophils in BAL fluid. Because Th2 responses were not induced by e.c. OVA exposure in Stat6(-/-) mice (mice lacking signal transducer and activator of transcription 6), the role of IL-13 was tested. In vivo depletion of IL-13 prevented Th2 responses induced by e.c. OVA exposure in IL-4(-/-) mice. These data demonstrate a marked difference in the IL-4 dependence of Th2 responses generated at two anatomic sites of natural allergen encounter and identify the skin as a particularly potent site for Th2 sensitization.

Stevens-Johnson syndrome limited to multiple sites of radiation therapy in a patient receiving phenobarbital.

Stevens-Johnson syndrome (SJS) is a severe cutaneous eruption that most often appears as an adverse reaction to a medication. There have been 21 reported cases of atypical erythema multiforme, toxic epidermal necrolysis, and SJS arising in patients receiving radiation therapy in addition to phenytoin, phenobarbital, or carbamazepine. We report the second case of SJS resulting from concomitant phenobarbital and radiation therapy, in which the eruption was limited to the sites of radiation, which were multiple.

Intracutaneous vaccination of rabbits with the E6 gene of cottontail rabbit papillomavirus provides partial protection against virus challenge.

DNA vaccination of rabbit skin with the L1 gene of cottontail rabbit papillomavirus (CRPV) has previously been shown to induce prophylactic immunity against CRPV. We now describe the effects of vaccination with the CRPV E6 gene, using the same approach. The experimental vaccine pdCMV-E6 encoded both the truncated and full length forms of CRPV E6 protein. The control vaccine pCMV-beta encoded beta galactosidase. Rabbits were vaccinated with DNA-coated gold particles, using a gene gun. Each rabbit received an initial vaccination with 30 micrograms DNA and 3 weeks later a booster vaccination, also with 30 micrograms DNA. pdCMV-E6-vaccinated rabbits developed E6-specific cellular immunity as determined by proliferation assays using peripheral blood mononuclear cells from animals prior to challenge, but did not develop detectable humoral immunity to E6 proteins, as evaluated by ELISA using two different E6 antigen preparations. Control rabbits developed humoral immunity to beta galactosidase. All rabbits were challenged by infection of nine skin sites with live CRPV virus and monitored for papilloma formation. None of four control rabbits was protected at any of the challenge sites. Of six rabbits vaccinated with pdCMV-E6, two were completely protected and one was virtually completely protected (tiny papillomas at just two of nine challenge sites). These three rabbits also exhibited significant E6-specific in vitro proliferative responses. The four E6 DNA-vaccinated rabbits that were not completely protected exhibited evidence of partial protection: some challenge sites did not form papillomas; papilloma onset was delayed; papilloma burden was less. These results demonstrate that partial prophylaxis against papillomavirus-induced disease can be achieved by intracutaneous vaccination with a recombinant plasmid encoding the papillomavirus.

Conservation of T cell receptor conformation in epidermal gammadelta cells with disrupted primary Vgamma gene usage.

A feature that distinguishes gammadelta T cell subsets from most alphabeta T cells and B cells is the association of expression of single T cell receptor (TCR) gamma and delta variable (V) region gene segments with specific anatomic sites. Mice lacking the TCR Vgamma5 chain normally expressed by most dendritic epidermal T cells were shown to retain a conformational determinant (idiotype) ordinarily expressed exclusively by such Vgamma5+ cells. Conservation by shuffled gammadelta TCR chains of an idiotype associated with a specific anatomic site indicates that for TCRgammadelta, as for immunoglobulin, conformation is associated to a greater extent with the function or development of lymphocyte repertoires than is the use of particular gene segments.

Intracutaneous vaccination of rabbits with the cottontail rabbit papillomavirus (CRPV) L1 gene protects against virus challenge .

A DNA vaccine encoding the major capsid protein L1 of cottontail rabbit papillomavirus (CRPV) was constructed and administered intracutaneously (i.c.) to rabbits as supercoiled plasmids bound to gold beads using a specialized delivery device ("gene gun"). L1 DNA-vaccinated rabbits developed cellular proliferative responses to CRPV virus-like particles and developed high titered antibodies with neutralizing activity to CRPV. Following experimental challenge with CRPV, all of the L1 DNA-vaccinated rabbits, vs none of the controls, were protected from papilloma formation. These results demonstrate that i.c. vaccination of rabbits with the L1 papillomavirus capsid gene can induce antibodies that protect against subsequent papillomavirus infection.

Disruption of epithelial gamma delta T cell repertoires by mutation of the Syk tyrosine kinase.

Chimeric mice in which lymphocytes are deficient in the Syk tyrosine kinase have been created. Compared with Syk-positive controls, mice with Syk -/- lymphocytes display substantial depletion of intraepithelial gamma delta T cells in the skin and gut, with developmental arrest occurring after antigen receptor gene rearrangement. In this dependence on Syk, subsets of intraepithelial gamma delta T cells are similar to B cells, but distinct from splenic gamma delta T cells that develop and expand in Syk-deficient mice. The characteristic associations of certain T-cell receptor V gamma/V delta gene rearrangements with specific epithelia are also disrupted by Syk deficiency.

Thy-1+ dendritic cells express truncated form of POMC mRNA.

The present study was designed to examine the expression of proopiomelanocortin (POMC) and its related derivative peptide adrenocorticotropic hormone (ACTH) in murine derived Thy-1+ dendritic cells. Immunostaining using a polyclonal antibody specific to ACTH and parent POMC molecule indicated the presence of POMC and its derivative peptide, ACTH, in cultures of Thy-1+ dendritic cells. To explore whether the POMC peptide is present as a reservoir or synthesized de novo in Thy-1+ dendritic cells. Northern blot analysis using 30-mer oligonucleotide probe for alpha-MSH/ACTH precursor POMC was carried out in total RNA from these cells. Northern blot analysis revealed the presence of POMC like mRNA transcript. However, the observed size of transcript was smaller (approximately 0.9 kb) than that expressed by murine AtT20 cells (approximately 1.2 kb), an anterior pituitary tumor cell line used as a positive control. These observations suggest that the epidermal Thy-1+ lymphocytes, like thymic lymphocytes, might serve the epidermis as one source for the synthesis of POMC. The synthesis and presence of POMC in the epidermis may be related to some of the pigmentary anomalies observed in many mucocutaneous disorders.

Immunobiology of mouse dendritic epidermal T cells: a decade later, some answers, but still more questions.

Over the past decade, overwhelming evidence has accumulated in many species, most notably in mice, that epithelial sites such as skin, intestine, and reproductive tract are populated with relatively discrete subsets of gamma delta cells. Such studies have identified several distinguishing and, in some cases, unique features of the dendritic epidermal T cells (DETC) populating the skin of all normal mice: homogeneous V5-J1-C gamma 1/V1-D2-J2-C delta T-cell receptors devoid of junctional diversity, apparent tissue restriction in adult mice to the skin, an important role for active hair growth in their localization and/or proliferation in the skin, and a capacity to recognize an antigen expressed on stressed epidermal cells. These properties have led to the hypothesis that DETC play distinctive roles in cutaneous immune surveillance and/or immunoregulation via recognition of a common self-antigen expressed by adjacent cells under various potentially harmful circumstances. Despite substantive advances in our knowledge about gamma delta cells in general (e.g., recent evidence that their manner of antigen recognition may be fundamentally different from that used by conventional alpha beta T cells) and about epithelial-specific subsets such as murine DETC in particular, it is clear that, compared with our understanding of alpha beta cells, major gaps still exist in our understanding of these cells. Persisting questions about DETC include: precise identification of the ligands for their homogenous T-cell receptors, the cellular and molecular requirements for their activation, their full range of functional activities, the reason(s) for the absence in normal human skin of a precise morphologic and phenotypic homologue, and, perhaps most important, their biologically relevant role(s) in cutaneous physiology, immunity, and/or pathology.

Murine epidermal V gamma 5/V delta 1-T-cell receptor+ T cells respond to B-cell lines and lipopolysaccharides.

The V gamma 5/V delta 1(+)-T-cell receptor (TCR)-bearing T-cell clone, 2CBET-3, was generated from C57BL/6 mice. Upon stimulation, 2CBET-3 cells produce interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha, but not IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, macrophage colony-stimulating factor, or interferon-gamma. These cells were evaluated for their ability to be stimulated by a variety of murine cell lines, including fibroblasts, trophoblasts, melanoma cells, embryonic carcinomas, B-cell lymphomas, mastocytoma cells, and keratinocytes. The human B-lymphoma cell line, Daudi, also was included in these studies. We found that 2CBET-3 cells produced cytokines up to several hundredfold above the control levels in response to the B-cell lines, Daudi, and A20/2J, but not to the B-cell line 439.4.2. After fixation with glutaraldehyde, Daudi and A20/2J continued to stimulate this gamma delta T-cell line. 2CBET-3 cells also responded to the keratinocyte line PAM212, but not to another, XB-2. When lipopolysaccharides (LPS) from Escherichia coli or S. typhimurium were added to 2CBET-3 cells in the presence of A20/2J cells, 2CBET-3 cells responded with increased cytokine production compared with the cytokine production in the presence of A20/2J cells alone. 2CBET-3 cells by themselves did not respond to LPS alone or to supernatants from A20/2J cells incubated with LPS. Unlike 2CBET-3, the epidermal T-cell hybridoma 70BET-49, expressing a V gamma 5/V delta 1-TCR identical to that of 2CBET-3, did not respond to A20/2J cells in the presence or absence of LPS, suggesting a requirement for molecules other than the TCR for V gamma 5/V delta 1-TCR+ T-cell stimulation by the B-cell lines and by LPS. This unique reactivity of gamma delta-TCR+ cells is different from that of alpha beta-TCR+ cells and may reflect a functional specialization of gamma delta-TCR+ cells in the response to bacterial infections.

Specific suppression of lupus-like graft-versus-host disease using extracorporeal photochemical attenuation of effector lymphocytes.

(C57BL/6 x DBA/2)F1 (B6D2F1) mice inoculated with parental DBA/2 (D2) splenocytes develop chronic stimulatory graft-versus-host reaction with many of the clinical manifestations of systemic lupus erythematosus. This investigation tested the ability of 8-methoxypsoralen (8-MOP) and ultraviolet A (UVA) light-treated D2 cells, primed to contain an expanded population of T cells specific for B6D2F1 major histocompatability complex antigens, to treat and/or prevent such systemic lupus erythematosus-like disease. 8-MOP/UVA-treated cells from B6D2F1-primed D2 donors were inoculated into B6D2F1 recipients weekly six to ten times, either before or after initiating graft-versus-host disease with normal D2 cells. A third group of B6D2F1 recipients were vaccinated weekly six times before disease initiation using 8-MOP/UVA-attenuated, B6D2F1-primed D2 cells that had been secondarily stimulated and expanded in vitro in the presence of irradiated B6D2F1 targets and interleukin-2. Control B6D2F1 mice were vaccinated with 8-MOP/UVA-treated D2 cells stimulated in vitro and/or in vivo with (C3H/HeJ x DBA/2)F1 cells. Only mice vaccinated with 8-MOP/UVA-attenuated D2-anti-B6D2F1 cells that were secondarily stimulated and expanded in vitro exhibited differences from controls when measured by the clinical parameters of ascites formation, and mean survival (p < 0.025). These groups also differed significantly in mean antinuclear antibody titer measured 14 weeks after disease initiation (p < 0.05). At 28 weeks, histologic evidence of systemic lupus erythematosus-like kidney disease was found only in the control group. These results indicate that photochemically attenuated D2-anti-B6D2F1 cells primed in vivo and secondarily stimulated and expanded in vitro are capable of vaccinating recipients against progression of graft-versus-host reaction-initiated systemic lupus erythematosus-like disease.

Superantigenic staphylococcal exotoxins induce T-cell proliferation in the presence of Langerhans cells or class II-bearing keratinocytes and stimulate keratinocytes to produce T-cell-activating cytokines.

Several staphylococcal toxins are among a growing number of immunostimulatory molecules called "superantigens" because of their ability, when presented by appropriate major histocompatibility complex class II+ accessory cells, to activate essentially all T cells bearing particular T-cell receptor V beta gene segments. We have examined the ability of murine epidermal Langerhans cells and/or keratinocytes to act as accessory cells in the T-cell response to the superantigens staphylococcal enterotoxin B and exfoliative toxin, also known as epidermolysin. Purified murine splenic T cells were stimulated with staphylococcal enterotoxin B or exfoliative toxin in the presence of Langerhans cells--enriched epidermal cells from normal mice or epidermal cells isolated from mice pretreated with recombinant interferon-gamma, a procedure that induces the expression of major histocompatibility complex class II molecules on keratinocytes. The data show that both Langerhans cells and class II-bearing keratinocytes can act as accessory cells in the T-cell response to staphylococcal enterotoxin B and exfoliative toxin. We also observed that both human and murine keratinocytes cultured in the presence of staphylococcal enterotoxin B or exfoliative toxin produce increased amounts of cytokine(s) capable of stimulating thymocytes and D10 cells, and that this toxin activity is independent of the level of expression of class II on keratinocytes. Studies by enzyme-linked immunosorbent assay showed that staphylococcal enterotoxin B stimulates keratinocytes to produce tumor necrosis factor-alpha but not interleukin-1, suggesting tumor necrosis factor-alpha and perhaps other cytokines are responsible for the T-cell proliferative activity. These results demonstrate that two distinct epidermal constituents (i.e. Langerhans cells and keratinocytes) can serve as accessory cells in the responses of T cells to superantigenic bacterial toxins. It is possible that such toxins contribute to the pathogenesis of a variety of skin diseases by either locally activating T cells bearing particular V beta genes and/or enhancing keratinocyte production of immunomodulatory cytokines.

The treatment of mastocytoma cells with 8-methoxypsoralen and long-wavelength ultraviolet radiation enhances cellular immunogenicity: preliminary results.

Evidence for the increased immunogenicity of mastocytoma cells (P815) treated with 8-methoxypsoralen (8-MOP) and long-wavelength ultraviolet radiation (UVA) is presented. A highly tumorigenic clone (P1) became much less tumorigenic (tum-) after repetitive phototreatments with 8-MOP (16 ng/mL) and UVA (1 J/cm2). The yield of tum- clones was proportional to the number of phototreatments. In a pilot study in which P1 cells were treated with three successive rounds of 8-MOP/UVA, one clone out of 73 was tum-. In a second series of experiments, the P1 cells were treated 10 times and 4 out of 100 clones were much less tumorigenic. When some of the tum- clones were administered intraperitoneally to DBA/2 mice, significant protection against challenge with the original P1 clone was observed. In addition, the transfer of immune cells from tum(-)-treated mice allowed the transfer of resistance to other tum- clones to immunosuppressed mice (650 rad). These results are consistent with earlier literature showing the potent mutagen, N-methyl-N'-nitrosoguanidine, led to mutations in P1 that altered the expression of new surface antigens, which stimulated the murine immune system such that there was also cross recognition of shared antigens on untreated P1 cells used to challenge the immunized mice. The increased immunogenicity that resulted from the less mutagenic 8-MOP/UVA treatment may arise by a similar mechanism and may be responsible in part for the efficacy of 8-MOP/UVA photochemotherapy for the treatment of cutaneous T cell lymphoma.

Idiopathic CD4+ lymphocyte deficiency. Report of an unusual case associated with atopic dermatitis and allergic contact dermatitis and review of the literature.

BACKGROUND: Idiopathic CD4+ lymphocyte deficiency is a newly described entity of apparently non-human immunodeficiency virus-associated helper T-cell depletion. The clinical spectrum continues to evolve, but, to date, it has included patients ranging from those with minimal symptoms to those who have died with acute opportunistic infections. Several individual cases have been previously reported, many with distinctive, primarily infectious, cutaneous manifestations. OBSERVATIONS: We describe a patient with idiopathic CD4+ lymphocyte deficiency distinguished by a unique clinical presentation of atopic dermatitis exacerbated by contact urticaria and allergic contact dermatitis. She has a persistent markedly diminished CD4+ lymphocyte count (absolute count less than 50), no serologic evidence of, or risk factors for, human immunodeficiency virus infection, and no history of opportunistic infections. CONCLUSIONS: We present a possible new cutaneous manifestation of idiopathic CD4+ lymphocyte disease and summarize the previously reported cases, with an emphasis on the cutaneous features.