Direct intralymphatic injection of peptide vaccines enhances immunogenicity.

Research to enhance the efficiency of vaccines focuses mainly on improving either the adjuvant or the type and form of the antigen. This study evaluates the influence of the administration route on the efficiency of a peptide-based vaccine. Peptide vaccines are generally administered subcutaneously or intradermally, from where they must reach secondary lymphatic organs to induce an immune response. We analyzed the efficacy of peptide vaccines administered directly into a lymph node. Using a MHC class I-binding peptide from lymphocytic choriomeningitis virus, we found that intralymphatic injection enhanced immunogenicity by as much as 10(6) times when compared to subcutaneous and intradermal vaccination. Intralymphatic administration induced CD8 T cell responses with strong cytotoxic activity and IFN-gamma production that conferred long-term protection against viral infections and tumors. These results should have immediate implications for clinical immunotherapy of infectious disease and cancer.

The interferon inhibiting cytokine IK is overexpressed in cutaneous T cell lymphoma derived tumor cells that fail to upregulate major histocompatibility complex class II upon interferon-gamma stimulation.

Cutaneous T cell lymphomas are characterized by an accumulation of malignant clonal lymphocytes in the skin and occasionally in the blood. We compared gene transcription profiles from cultured clonal lymphocytes with autologous healthy blood lymphocytes by microarray hybridization. Cutaneous T cell lymphoma derived cells transcribed high amounts of an interferon inhibiting cytokine factor. The presence of an interferon inhibiting cytokine factor was confirmed in 12 skin biopsies of mycosis fungoides and Sézary syndrome derived blood lymphocytes by reverse transcriptase-polymerase chain reaction. The presence of interferon inhibiting cytokine factor mRNA in Sézary syndrome derived lymphocytes was associated with a lack of HLA class II upregulation after stimulation with interferon-alpha and interferon-gamma. This was not due to a loss of the interferon signaling cascade as the presence of interferon-signaling components was confirmed by reverse transcriptase--polymerase chain reaction on the transcriptional level. The elevated constitutive interferon inhibiting cytokine factor expression observed in cutaneous T cell lymphoma derived cells was insensitive to interferon-gamma stimulation, but was enhanced in normal peripheral blood mononuclear cells. We suggest that interferon inhibiting cytokine factor contributes to the lack of HLA class II upregulation in lymphoma cells. Interferon inhibiting cytokine factor may participate in providing a microenvironment at the tumor site insensitive to interferon-gamma stimulation and thus prevents an efficient local immune response.

Coexpression of CD40 and CD40 ligand in cutaneous T-cell lymphoma (mycosis fungoides).

Microarray analysis is a promising new approach for creating specific expression profiles of multiple genes simultaneously. We quantitatively analyzed differential gene expression patterns in mycosis fungoides-derived clonal T cells and autologous, identically cultured CD4+ lymphocytes using microarrays containing 588 cDNA segments from genes relevant to cell signaling, carcinogenesis, and apoptosis. Among other dissimilarities, neoplastic T cells showed coexpression of CD40 (Bp50) and CD40 ligand (gp39, CD154). These results could be corroborated by reverse transcription-PCR, immunohistochemistry, and two-color immunofluorescence staining. Our data suggest that in cutaneous T-cell lymphoma, CD40/CD40 ligand interactions might represent a paracrine loop that is crucial not only in preventing apoptosis or positively regulating growth but also in homing of neoplastic cells to the skin.

Analysis of tumor-infiltrating lymphocytes in cutaneous squamous cell carcinoma.

OBJECTIVE: To characterize tumor-infiltrating lymphocytes (TILs) within lesions of cutaneous squamous cell carcinoma (SCC) and related disorders. DESIGN: Case series with 1- and 2-color immunohistologic, molecular biological analysis of T-cell clonality and in vitro cytotoxicity assays. SETTING: Academic medical center. PATIENTS: Twenty-one patients, including 6 with actinic keratoses, 4 with SCC in situ, and 11 with invasive SCC. RESULTS: CD8+ TILs were present within lesions of cutaneous SCC and AK. These cells constituted a variable minority of the total T-cell infiltrate, and many expressed a phenotype consistent with major histocompatibility complex-restricted cytotoxic T lymphocytes: CD3+, TIA1+, CD16-, CD56-, CD57-. They also expressed HLA-DR, suggesting their activation in vivo. Virtually all T cells were T-cell receptor (TCR)-beta + delta, indicating that they expressed the TCR-alpha beta protein heterodimer. Molecular biological analysis of TCR-gamma gene rearrangements by the polymerase chain reaction and denaturing gradient gel electrophoresis technique indicated that the TILs were polyclonal. Functional studies suggested that TILs derived from SCC lesions were cytotoxic for autologous tumor cell targets. CONCLUSION: Tumor-infiltrating lymphocytes within cutaneous SCC lesions contain a subpopulation of polyclonal, major histocompatibility complex-restricted cytotoxic T lymphocytes expressing the TCR-alpha beta heterodimer.

Induction of biologically active IL-1 beta-converting enzyme and mature IL-1 beta in human keratinocytes by inflammatory and immunologic stimuli.

IL-1beta, a major mediator of inflammatory and immunologic skin disease, undergoes post-translational site-specific cleavage by a novel cysteine protease termed IL-1beta-converting enzyme (ICE). Although in human skin keratinocytes produce significant amounts of the 31-kDa IL-1beta precursor protein, they fail under nonpathologic conditions to convert it to the 17.5-kDa bioactive form. In this study, we examined whether haptens and inflammatory agents might serve as stimuli for ICE activity in human keratinocytes, and, if so, whether ICE activity might precipitate enzymatic processing of IL-1beta to its 17.5-kDa form. Baseline levels of ICE mRNA were detected in keratinocyte cultures devoid of Langerhans cells and were up-regulated by nontoxic concentrations of the reactive hapten urushiol and by the irritant chemicals sodium lauryl sulfate and PMA. Although untreated keratinocytes expressed the 31-kDa form of the protein, 17.5-kDa IL-1beta was easily detected in keratinocytes and keratinocyte supernatants treated with either urushiol or the irritant chemicals. Enzymatic conversion from the 31-kDa to the 17.5-kDa form of IL-1beta was blocked by addition of a highly specific aldehyde inhibitor that contained a tetrapeptide recognition sequence specific for ICE, but not by an aldehyde inhibitor of a related ICE-like cysteine protease. Induction of IL-1beta-converting enzyme by immunologic and inflammatory stimuli may be one of the key regulatory elements in the pathogenesis of allergic and irritant contact hypersensitivity.

Major histocompatibility complex class I molecule serves as a ligand for presentation of the superantigen staphylococcal enterotoxin B to T cells.

Superantigens, such as staphylococcal enterotoxin B (SEB), elicit a strong proliferative response in T cells when presented in the context of major histocompatibility complex (MHC) class II molecules. We observed a similar T-cell response, when MHC class II-negative epidermal cell lines were employed as antigen-presenting cells. Immunoprecipitation studies indicated that the ligand to which SEB bound had a molecular mass of 46 kDa. Radiolabeled SEB could be immunoprecipitated from isolated membrane proteins on the SCC13 epidermal cell line with a monoclonal antibody directed against the MHC class I molecule, and transfection of the K-562 cell line with MHC class I molecules showed a 75% increased SEB-binding capacity compared with the nontransfected MHC class I- and class II-negative counterpart. In functional studies, antibodies to the MHC class I molecule inhibited T-cell proliferation by at least 50%. From these studies, we conclude that MHC class I molecules on malignant squamous cell carcinomas serve as ligands for SEB, which, given the appropriate costimulatory signals, is sufficient to allow for superantigen-induced T-cell proliferation.

Reduced growth factor requirements and accelerated cell-cycle kinetics in adult human melanocytes transformed with SV40 large T antigen.

Melanomas develop with high frequency in transgenic mice in which oncogenic sequences of the SV40 DNA tumor virus have been specifically targeted to melanocytes. To investigate the role of SV40 in melanomagenesis, cultured human melanocytes were transformed with a retroviral shuttle vector encoding the SV40 large T antigen and examined for changes in cell-cycle kinetics and growth-factor dependence. Colonies expressing the viral oncogene were morphologically indistinguishable from their non-T-antigen-transformed counterparts. Also like normal melanocytes, the infected cells remained anchorage dependent and non-tumorigenic in nude mice. However, T-antigen-positive cultures exhibited significantly accelerated population doubling times, increased saturation densities with highly confluent monolayers and a three- to fourfold extended life span. Most interestingly, cell-cycle analysis revealed a measurable shift from quiescent to cycling cells in T-antigen-expressing cultures and an acquired ability to progress more rapidly through G1. Moreover, T-antigen-positive melanocytes proliferated in the absence of PMA and required markedly reduced levels of exogenous bFGF. These studies indicate that the viral oncogen of simian virus 40 provides melanocytes with distinct growth advantages that may render these cells unusually susceptible to additional environmental challenges necessary for full expression of the malignant phenotype.

Differentiation and clonality of lesional lymphocytes in small plaque parapsoriasis.

BACKGROUND: Small plaque parapsoriasis is an idiopathic chronic dermatosis characterized by patches on the trunk and extremities that are often smaller than 5 cm in diameter and that sometimes have a digitate contour. These latter cases are often referred to as digitate dermatosis. Histopathologic examination reveals a mild superficial perivascular lymphocytic infiltrate associated with mild spongiosis and parakeratosis. To characterize this disease more completely, we analyzed the differentiation and clonality of lesional lymphocytes using immunohistologic and molecular biologic methods. OBSERVATIONS: We studied five cases using a frozen-section immunoperoxidase technique. In each case, there was a predominantly CD4+ T-cell infiltrate admixed with CD8+ T cells, Langerhans cells/indeterminate cells, and macrophages. In three cases, the clonality of lesional T cells was studied by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified T-cell receptor-gamma gene rearrangements. Two cases showed a dominant clonal pattern, while one case exhibited a polyclonal pattern. Clinical follow-up disclosed persistent disease in one of the two clonal cases, while lesions in the other clonal case and the polyclonal case gradually resolved. CONCLUSIONS: Our findings indicate that small plaque parapsoriasis is a clinically indolent, histopathologically nonspecific, predominantly CD4+ T-cell-mediated disease that, at least in some cases, contains a dominant T-cell clone. These features put small plaque parapsoriasis into a category with certain other members of the parapsoriasis group, namely, pityriasis lichenoides and lymphomatoid papulosis, which have been shown to be clonal T-cell disorders despite their clinically benign course. It remains to be determined if the dominant T-cell clones identified in some cases of small plaque parapsoriasis can ever be the direct precursors of overt cutaneous T-cell lymphomas.