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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.

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.

The Dark Side of Langerhans Cells Several immune cell populations reside in the skin and are thought to provide a protective barrier against infections and to act as sentinels against malignant transformation. However, studies in mice that lack Langerhans cells, a subset of dendritic cells, have suggested that these cells may actually promote tumorigenesis. Using a mouse model of squamous cell carcinoma, Modi et al. (p. 104 ) now reveal how Langerhans cells may promote the transformation of skin epithelial cells. In response to the carcinogen 7,12-dimethylbenz[α]anthracene (DMBA), Langerhans cells increased their expression of the cytochrome P-450 enzyme CYP1B1, which can metabolize DMBA to the mutagenic DMBA- trans -3,4-diol. Thus, besides their functions in regulating the adaptive immune response, Langerhans cells may participate in the metabolism of environmental carcinogens.

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.

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.

Intraepithelial lymphocytes constitute a group of T cells that express mainly monospecific or oligoclonal T cell receptors (TCRs). Like adaptive TCR alphabeta+ T cells, intraepithelial lymphocytes, a subset enriched in TCR gammadelta+ T cells, are proposed to be positively selected by thymically expressed self agonists, yet no direct evidence for this exists at present. Mouse dendritic epidermal T cells are prototypic intraepithelial lymphocytes, displaying an almost monoclonal TCR gammadelta+ repertoire. Here we describe an FVB substrain of mice in which this repertoire was uniquely depleted, resulting in cutaneous pathology. This phenotype was due to failure of dendritic epidermal T cell progenitors to mature because of a heritable defect in a dominant gene used by the thymic stroma to 'educate' the natural, skin-associated intraepithelial lymphocyte repertoire to be of physiological use.

Upregulation of the inducible gene products MICA (human) and Rae-1 (mouse) may promote tumor surveillance and autoimmunity by engaging the activating receptor NKG2D on natural killer (NK) cells and T cells. Nevertheless, sustained expression of MICA by tumors can also elicit NKG2D downregulation, perhaps indicating 'immunoevasion'. Investigating this paradox, we report here that constitutive Rae-1epsilon transgene expression in normal epithelium elicited local and systemic NKG2D downregulation, generalized but reversible defects in NK cell-mediated cytotoxicity and mild CD8(+) T cell defects. The extent of NKG2D downregulation correlated well with the incidence and progression of cutaneous carcinogenesis, emphasizing the utility of NKG2D as a marker of tumor resistance. Thus, NKG2D engagement is a natural mediator of immunosurveillance, which can be compromised by locally sustained ligand expression but potentially restored by innate immune activation.

An adverse cutaneous reaction to a systemically administered drug may rarely manifest as acute generalized exanthematous pustulosis (AGEP). Several recent reports have documented positive patch test results in patients with a history of AGEP, while two have demonstrated drug-specific in vitro lymphocyte proliferative responses. These findings suggest that drug-specific T cells mediate AGEP. We describe two patients with a history of AGEP who each demonstrated positive patch test results specific for the inciting drug: Patient #1 to the antibiotic metronidazole, and Patient #2 to the calcium channel-blocker diltiazem. Histologic examination of biopsy specimens taken from the patch test sites of these patients revealed spongiotic dermatitis and perivascular lymphocytes consistent with a delayed-type hypersensitivity reaction, rather than demonstrating subcorneal neutrophilic pustules more typical of AGEP. In vitro testing by measuring peripheral T cell proliferative responses to chemically purified drug correlated with the clinical response. In a direct cross-comparison, patch test results were shown to correlate with in vitro lymphocyte proliferative responses in two patients with a history of AGEP to different drugs. These findings provide additional evidence that the pathogenesis of AGEP involves a T cell-mediated immune response.

Studies in an in vitro model of cutaneous T-cell lymphoma (CTCL) demonstrated that CTCL cell proliferation is stimulated by direct contact with autologous, immature dendritic cells (DCs), suggesting that CD4+ CTCL cell division is driven by antigens presented by DC major histocompatibility complex (MHC) class 2. We now report that the T-cell receptor (TCR) of the CD4+ CTCL cells is triggered after interaction with DCs loaded with apoptotic CTCL cells, as shown by reduced membrane expression of CD3 and the TCR, up-regulation of cytotoxic T lymphocyte antigen-4 (CTLA-4), and calcium mobilization. CTCL cells adopt a T-regulatory (Treg) phenotype expressing CD25/CTLA-4 and FoxP3 and secreting interleukin-10 (IL-10) and transforming growth factor-β (TGF-β). Treg CTCL cells suppress normal T-cell antigen-driven secretion of IL-2 and interferon-γ (IFN-γ). Blocking DC MHC class 2 expression or transport inhibited CTCL cell adoption of a Treg phenotype. Allogeneic CTCL cells or normal CD4 T cells served as sources of apoptotic material for CTCL cell conversion to a Treg phenotype. Conversion of CTCL cells to Treg cells may explain the anergic, immunosuppressive nature of the malignancy. (Blood. 2005;105:1640-1647)

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.

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.

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.

The intraepithelial lymphocyte (IEL) network of T-cell receptor gammadelta+ (Vgamma5+) dendritic epidermal T cells (DETC) in murine skin down-regulates cutaneous inflammation, although the mechanism is unknown. Thymosin-beta4 (Tbeta4), identified by serial analysis of gene expression as a predominant transcript in gut IEL, encodes both a ubiquitous actin-binding protein (UTbeta4) with demonstrated capacity to inhibit neutrophilic infiltration, and a splice-variant limited to lymphoid tissue (LTbeta4) with unknown bioactivity. Freshly isolated Vgamma5+ DETCs expressed both forms, while only LTbeta4 was preferentially up-regulated after cellular activation in vitro. To compare the anti-inflammatory properties of LTbeta4 and UTbeta4 in the skin in vivo, the biological activities of synthesized polypeptides were assessed using three different strategies: neutrophil infiltration by footpad lambda-carrageenan injection; irritant contact dermatitis to 12-O-tetradecanoylphorbol 13-acetate; and allergic contact dermatitis to 2,4-dinitrofluorobenzene. These studies clearly showed that the anti-inflammatory activities of LTbeta4 were broader and most often stronger than those of UTbeta4. Thus, the activation-responsive expression of the lymph-specific form of Tbeta4 may be one mechanism by which DETC, and possibly other IELs, down-regulate local inflammation.

TCRgammadelta-transgenic IL-7(-/-) mice were generated to determine whether T cells containing productively rearranged TCRgammadelta genes have additional requirements for IL-7 within the thymus or peripheral lymphoid tissues. Differences in developmental requirements for IL-7 by TCRgammadelta cells were noted and were linked to derivation from fetal- vs adult-type precursors in the thymus. Although TCRgammadelta cells are absent from IL-7(-/-) mice, TCRgammadelta cells were restored to the thymus and periphery by expression of TCRgammadelta transgenes. Endogenous TCRgamma chains were expressed by IL-7(+/-) but not IL-7(-/-) TCRgammadelta-transgenic mice, providing direct support for findings that IL-7 is necessary for rearrangement and expression of TCRgamma genes. The number of TCRgammadelta thymocytes was 10-fold reduced in TCRgammadelta-transgenic IL-7(-/-) embryos; however, adult TCRgammadelta-transgenic IL-7(-/-) or IL-7(+/-) mice had similar numbers of fetal thymus-derived TCRgammadelta cells in their skin. Thus, fetal TCRgammadelta cells required IL-7 for TCR rearrangement, but not for proliferation or survival in the periphery. In contrast, the numbers of TCRgammadelta cells in other tissues of TCRgammadelta-transgenic IL-7(-/-) mice were not completely restored. Moreover, coincident with the transition from the first to second wave of T cell precursors maturing in neonatal thymus, thymus cellularity of TCRgammadelta-transgenic IL-7(-/-) mice dropped significantly. These data indicated that in addition to TCRVgamma gene rearrangement, TCRgammadelta cells differentiating from late fetal liver or adult bone marrow precursors have additional requirements for IL-7. BrdU incorporation studies indicated that although IL-7 was not required for TCRgammadelta cell proliferation, it was required to prolong the life span of mature TCRgammadelta 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 γδ T-cell receptor (TCR)+ subset of mouse skin. In this article, we look at immunoregulatory γδ T cells, and the growing evidence for tissue-associated immunoregulation mediated by both γδ T cells and αβ T cells.

Dynamic imaging portrays intraepidermal T cells as truly autoreactive, constitutively transmitting signals from a putative ligand expressed by normal epithelial cells.

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.

Acute generalized exanthematous pustulosis (AGEP) is an acute sterile pustular eruption most commonly induced by medications. We present a case of AGEP with erythroderma following use of midodrine in a 58-year-old man. Although antibiotics are most commonly implicated in AGEP, we emphasize that nonantibiotic agents also may cause AGEP, which often manifests after a longer time interval compared to antibiotic-associated AGEP.

The localization of γδ T cells within epithelia suggests that these cells may contribute to the down-regulation of epithelial malignancies. We report that mice lacking γδ 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+γδ 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.

A preferred anti-cancer vaccine would be tumor-specific, simple to rapidly construct and safe to administer. It would permit immunization against a spectrum of the tumor's distinctive antigens, without requiring their prior identification. Toward these goals, we describe a modification of standard extracorporeal photopheresis (ECP) which initiates, within a single day, both monocyte-to-dendritic cell (DC) differentiation and malignant cell apoptosis. The transition of monocytes to immature DCs was identified by the expression of cytoplasmic CD83 and membrane CD36 in the absence of membrane CD14 staining, as well as induction of membrane CD83 expression. Differentiating DCs were avidly phagocytic and engulfed apoptotic malignant T cells. Differentiating DCs were capable of stimulating significant proliferation of normal alloreactive lymphocyte responders, indicting increased expression of membrane MHC class II molecules. This approach provides a clinically practical means of developing tumor-loaded cells that have initiated the transition to DCs without the requirement of exogenous cytokines, excessive cellular manipulation or isolation. Construction of DC vaccines using this methodology can be generalized to other diseases and may offer a novel approach for improved cancer immunotherapy.

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.

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.

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 (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.

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.

A feature that distinguishes γδ T cell subsets from most αβ T cells and B cells is the association of expression of single T cell receptor (TCR) γ and δ variable (V) region gene segments with specific anatomic sites. Mice lacking the TCR Vγ5 chain normally expressed by most dendritic epidermal T cells were shown to retain a conformational determinant (idiotype) ordinarily expressed exclusively by such Vγ5+cells. Conservation by shuffled γδ TCR chains of an idiotype associated with a specific anatomic site indicates that for TCRγδ, 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.

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.

Summary 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 αVβ3 and α5β1integrin 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.

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.

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.

(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 (p0.025). These groups also differed significantly in mean antinuclear antibody titer measured 14 weeks after disease initiation (p0.05). 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.

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.

αβ 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.

The concept of skin-associated lymphoid tissue embraces those cells and functions that are integrated in the cutaneous host defense. Recently, it has been possible to identify those circulating T-cells that are skin associated. These cells display the cell-surface phenotype of memory T cells (CD45RO+) and express the cutaneous lymphocyte antigen, a tissue-selective homing receptor involved in directing T-cell traffic to inflamed skin. To investigate the participation of this skin-associated T-cell subset in the pathogenesis of cutaneous T-cell lymphoma, we studied 16 patients with erythrodermic cutaneous T-ceIl lymphoma for the presence of these surface proteins on circulating cells. Results were compared with eight patients in remission and eight with minimal patch-plaque cutaneous T-cell lymphoma. The mean expression of both CD45RO and cutaneous lymphocyte antigen were significantly greater in the erythrodermic patients than in the other two patient groups. Expression of these markers was shown to be on the cells of the malignant clone by two-color flow cytometry. These results demonstrate that the malignant cells of cutaneous T-cell lymphoma express the markers of skin homing lymphocytes and that their levels are increased in the erythrodermic cutaneous T-cell lymphoma patients. Moreover, the findings suggest a critical role for the skin-selective homing receptor cutaneous lymphocyte antigen in the pathogenesis of cutaneous T-cell lymphoma.

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.

Background Palifermin is a recombinant human keratinocyte growth factor that is used to reduce the duration and severity of oral mucositis in patients undergoing hematopoietic stem cell transplantation after myelotoxic therapy. Cutaneous adverse reactions associated with keratinocyte growth factor are reported to be rash, pruritus, and erythema. Observations After receiving palifermin following autologous hematopoietic stem cell transplantation and treatment with melphalan, a patient developed erythema and lichenoid papules that were distributed primarily in intertriginous areas. A biopsy specimen of the papules showed a striking resemblance to verrucae, but in situ hybridization studies were negative for human papillomavirus. Immunohistochemical staining with antibodies to Ki-67 and cytokeratin 5/6 showed increased keratinocyte proliferation in lesional skin. Conclusions After treatment with palifermin, a papular eruption clinically resembling lichen planus or plane warts, with histologic features of verruca plana, and intertriginous erythema may occur. In this case, neither eruption required treatment, and spontaneous resolution was observed over days to weeks. Histopathologic staining patterns of Ki-67 and cytokeratin 5/6 may be useful in identifying adverse reactions to palifermin therapy.

Short-term bulk cultures and some long-term clones and lines of murine T cell receptor (TcR) gamma/delta-bearing epidermal T cells (dEC) were found to express an Fc gamma receptor II (Fc gamma RII), as revealed by reactivity with the monoclonal antibody 2.4G2. Northern blot analysis showed that the Fc gamma RII expressed on dEC is encoded solely by the Fc gamma R alpha gene. While all the various cultured dEC cell populations analyzed exhibit lectin-dependent cellular cytotoxicity, only those which expressed Fc gamma R alpha were also capable of mediating antibody-dependent cellular cytotoxicity (ADCC). These results in combination with the previous demonstration of Fc gamma R alpha on mouse natural killer cells support an essential role for Fc gamma R alpha in ADCC and extend an analogy with surface CD16 (Fc gamma RIII) expression and ADCC in human natural killer cells and peripheral TcR gamma/delta T cells.

Binding of antigen-presenting cells (APC) to T cells via adhesion molecules is thought to deliver accessory signals that are required for efficient T-cell activation. To determine whether Langerhans cells (LC) express relevant adhesion molecules on their surfaces, we employed two-color immunofluorescence. Human epidermal cells (EC), Ficoll-enriched for LC (greater than 10%), were incubated with monoclonal antibodies (MoAb) specific for the adhesion molecules CD11a (LFA-1 alpha), CD18 (LFA-1 beta), or ICAM-1; staining was evaluated by fluorescence microscopy. After 12 h of culture only HLA-DR+ cells (LC) expressed CD11a, CD18, and ICAM-1. As a test for the functional relevance of such adhesion molecule expression, we examined the capacity of the above MoAb to block LC stimulation of alloreactive T cells: EC were co-cultured with allogeneic peripheral blood mononuclear leukocytes (PBML) for 5 d in the presence or absence of MoAb; proliferation was measured by [3H]-thymidine uptake. MoAb against CD11a, CD18, or ICAM-1 reduced the allostimulatory capacity of LC by greater than 70%; combinations of these MoAb reduced proliferation even more (90%). We conclude that interaction of adhesion molecules on LC with ligands on T cells is required for optimal allo-antigen-dependent T-cell activation, perhaps by delivering accessory signals.

B cells, alphabeta T cells and gammadelta T cells are conserved lymphocyte subtypes encoding their antigen receptors from somatically rearranged genes. alphabeta T cells undergo positive selection in the thymus by engagement of their T cell receptors (TCRs) with self-peptides presented by major histocompatibility complex molecules. The molecules that select gammadelta T cells are unknown. Vgamma5+Vdelta1+ cells comprise 90% of mouse epidermal gammadelta T cells. By mapping and genetic complementation using a strain showing loss of Vgamma5+Vdelta1+ cells due to a failure of thymic selection, we show that this defect is caused by mutation in Skint1, a newly identified gene expressed in thymus and skin that encodes a protein with immunoglobulin-like and transmembrane domains. Skint1 is the prototypic member of a rapidly evolving family of at least 11 genes in mouse, with greatest similarity to the butyrophilin genes. These findings define a new family of proteins mediating key epithelial-immune interactions.

The epidermis of all strains of normal mice is populated by two distinct dendritic, bone marrow-derived cells: Langerhans cells and CD4-CD8- Thy-1+ dendritic epidermal T cells (DETC). The overwhelming majority of DETC are an unusually homogeneous population of thymic-dependent cells which express CD3-associated T-cell antigen receptors (TCRs) of the gamma/delta type, thereby distinguishing them from conventional CD4+CD8- or CD4-CD8+ T cells expressing CD3-associated alpha/beta TCR. Most DETC are ontogenetically primitive, derived from early fetal thymocytes with a preferential, but poorly understood tropism for the epidermis. Like the TCR on other populations of gamma/delta cells, which preferentially populate other epithelia such as in the gut and lung, the TCR on most DETC selectively utilize particular variable (V) gene segments (i.e., V gamma 3 and V delta 1 for DETC vs V gamma 5 and V delta 4 or V delta 6 for intestinal intraepithelial lymphocytes). However, unlike other gamma/delta populations whose TCR junctional regions exhibit marked heterogeneity, DETC junctional diversity is extremely limited. This lack of TCR heterogeneity among DETC suggests they recognize a narrow range of physiologic ligands (antigens) and that this recognition is restricted not by conventional polymorphic class-I or class-II MHC molecules, but rather by relatively nonpolymorphic self MHC-like molecules of the class Ib MHC type [e.g., Qa, TL, and CD1 (T6)]. Additional studies are required to clarify precisely what DETC recognize, their relevant biological functions, as well as their relationship(s) to the gamma/delta cells recently identified in human skin.

Ultraviolet radiation (UVR)3 from within the spectrum B (UVB) has the capacity to distort the induction of contact hypersensitivity (CH) in murine skin. A damaging effect of UVB on epidermal Langerhans cells (LC) appears to be universal in all genetically defined strains of mice tested. However, while UVB impairs the induction of CH in some strains of mice, it has no apparent effect on CH in others. Thus, a disparity exists between the effects of UVB on LC and on CH. This is a paradox because LC are generally regarded to serve as the antigen-presenting cells of the skin, placing them at the earliest stages of induction of CH. One possible explanation for this paradox has been that UVB-susceptible strains of mice may generate hapten-specific suppressor T cells, whereas their UVB-resistant counterparts may not, when their skin is treated with UVR and painted with haptens such as dinitrofluorobenzene (DNFB). This possibility was excluded by examining the capacity of UVR and hapten to generate suppressor T cells in several different inbred strains of mice. The results indicate that the induction of hapten-specific afferent T suppressor cells is a universal sequela to treatment of mice with UVB and hapten, irrespective of whether the mice display the phenotype of vigorous CH or not. Thus, the genetic basis of UVB-resistance does not reside in the ability of UVR to induce suppressor T cells. Rather, attention should now be focused on its ability to interrupt induction of effector mechanisms.

There is a longstanding but poorly understood epidemiologic link between inflammation and cancer. Consistent with this, we previously showed that αβ T cell deficiency can increase resistance to chemical carcinogenesis initiated by 7,12-dimethylbenz[ a ]anthracene and promoted by phorbol 12-myristate 13-acetate. This provoked the hypothesis that αβ T cell deficiency removed T regulatory cells that limit the anti-tumor response or removed a specific tumor-promoting (T-pro) T cell population. Here we provide evidence for the latter, identifying a novel CD8 + subset that is a candidate for T-pro cells. We demonstrate that CD8 cell-deficient mice show substantially less tumor incidence and progression to carcinoma, whereas susceptibility is restored by CD8 + cell reconstitution. To characterize the putative T-pro cells, tumor-infiltrating lymphocytes were isolated from normal and CD4 −/− mice, revealing an activated population of T cell receptor αβ + CD8 + CD44 + CD62L − cells expressing the inflammatory mediators IFNγ, TNFα, and cyclooxygenase-2, but deficient in perforin, relative to recirculating cells of equivalent phenotype. This novel population of CD8 + T cells has intriguing similarities with other lymphocytes that have been associated with tissue growth and invasiveness and has implications for inflammation-associated carcinogenesis, models of cancer immunosurveillance, and immunotherapeutic strategies.

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.

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.

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.

Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature T lymphocytes, most of which express alpha/beta type T-cell receptors (TCRs). The cause of CTCL is unknown, but hypotheses postulating chronic stimulation of TCRs by superantigen or by a leukemogenic virus have been proposed. Either mechanism might produce bias in the TCR variable (V) region types used by the malignant cells. To determine if TCR alpha use is restricted in CTCL, we used reverse transcription and the polymerase chain reaction to determine V alpha and V beta usage by malignant cells purified from the peripheral blood of leukemic patients with CTCL. Usage of alpha chain V region segments appeared totally random; malignant lymphocytes isolated from each of six patients used different V alpha regions. As has been previously reported, no bias was found in beta chain V region usage either. In addition to productive (in frame) TCR V region mRNAs in malignant cells from each patient, we detected non-productive (out of frame) beta chain transcripts in these cells in two of six patients, and non-productive alpha chain transcripts in five of six. Residual normal peripheral blood lymphocytes from these patients showed a random, polyclonal or oligoclonal pattern of V region usage. We conclude that there is no bias in V region usage in CTCL, making it unlikely that interactions between superantigen or virus and the TCR V regions play a role in the pathogenesis of CTCL.

Although T cell receptor (TCR)gammadelta+ and TCRalphabeta+ cells are commonly viewed as functionally independent, their relatedness and potential interdependence remain enigmatic. Here we have identified a gene profile that distinguishes mouse gammadelta cell populations from conventional alphabeta T cells. However, this profile was also expressed by sets of unconventional alphabeta T cells. Therefore, whereas TCR specificity determines the involvement of a T cell in an immune response, the cell's functional potential, as assessed by gene expression, does not segregate with the TCR. By monitoring the described gene profile, we show that gammadelta T cell development and function in TCRbeta-deficient mice was impaired because of the absence of alphabeta T cell progenitors. Thus, normal gammadelta cell development is dependent on the development of conventional alphabeta T cells.