Light dosimetry using the P3 approximation.

In earlier work, we demonstrated that radiance, calculated using the P3 approximation in a plane wave geometry, could be used to accurately predict the optical parameters of an Intralipid/methylene blue phantom. Plane wave geometry is impractical for clinical use but the results of this work encouraged us to further develop the P3 approximation for a spherical geometry, described in this paper. Radiance predicted by this model for a defined Intralipid/methylene blue phantom was compared with radiance measured in this phantom. The results demonstrate that the spherical derivation of the P3 approximation will reproducibly predict optical parameters of a tissue phantom as effectively as the slab geometry derivation of the P3 approximation. In a similar protocol, the P3 approximation was used to estimate the optical parameters of ex vivo human prostate. Radiance in this case was measured in the prostate samples using an after loading technique. Three prostate samples tested were found to be surprisingly optically homogeneous. The after loading protocol described in this paper could form the basis of a minimally invasive and effective clinical method to optically characterize human prostate.

Targeting of phototoxic drugs to antigen-specific T lymphocytes in vitro using antigen-presenting cell membranes.

We have used the complex of antigen with class II major histocompatibility proteins (Ia) in membrane-bound form to target a phototoxic compound to antigen-specific T cell hybridomas in vitro. The iodoacetamidyl ester of phototoxic pyrene was bound covalently to antigen-presenting cells (APC), and protein antigens were added to the cells for processing, presentation and targeting of the drug to three different T hybridomas specific for myelin basic protein (MBP), ovalbumin (OVA) and keyhole limpet hemocyanin (KLH). The B hybridoma LS102.9 was used as APC to present MBP, KLH and either a tryptic digest of OVA or the synthetic peptide OVA323-339 to these T cells. A transformed B lymphoma, which expresses trinitrophenol (TNP)-specific surface IgM, A20-HL, was used to present TNP conjugates of KLH and OVA to T cells. Either the antigen-bearing intact APC or Ia+ membranes shed spontaneously from them were used as drug carriers to target pyrene to the T cells. In the dark, or in the absence of pyrene, both the intact APC or the shed membranes stimulated interleukin-2 (IL-2) production by the T cells in an antigen-specific way. After UVA (320-400 nm) irradiation, both forms of these drug carriers had an antigen-specific toxic effect on the T hybridoma cells with receptors for the antigen that they carried. Both spontaneous T cell proliferation and antigen-induced IL-2 production were inhibited. The shed membranes had a more antigen-specific toxic effect than the intact APC, which tend to settle out with the T cells in the microtiter plates, possibly causing nonspecific contact.(ABSTRACT TRUNCATED AT 250 WORDS)

Major histocompatibility complex class II-restricted cytotoxicity by self-myelin basic protein-reactive T-cell hybridomas: evidence for a tumour necrosis factor-independent nucleolytic mechanism.

Direct cytotoxicity by class II-restricted T cells has been proposed as a potential mechanism in autoimmune tissue damage, as well as in immunoregulation. We used I-A(s)-restricted non-granular cytotoxic T-cell hybridomas (BP24.29 and BP47.7), specific for self-determinants on myelin basic protein (MBP), and different monoclonal targets, in order to characterize the mechanism of killing used by these cells. An early lesion at the level of the target cell nucleus was indicated by the fact that target DNA lysis ([3H]thymidine release) proceeded 2-2.5-fold as rapidly as cytoplasmic lysis (51Cr release) over the first 14 hr after stimulation. Cytotoxicity was relatively resistant to inhibition by anti-calcium agents (TMB-8 and verapamil), even under conditions which blocked interleukin-2 (IL-2) release. Although tumour necrosis factor (TNF) has been proposed as one mediator of class II-restricted cytotoxicity, these cells (i) released no detectable TNF after stimulation with antigen, concanavalin A (Con A), or anti-CD3, (ii) readily lysed TNF-resistant targets (A20 and LS-102.9), and (iii) had no cytotoxic effect on TNF-sensitive cells (L929). Substantial 'bystander' killing of I-A-mismatched targets was observed, which was 13-37% of the cognate (I-A(s)-restricted) cytotoxicity measured in parallel. This finding may indicate an effector mechanism in autoimmune demyelination, since the myelin-forming oligodendrocytes of the central nervous system are not inducible for major histocompatibility complex (MHC) class II expression.

Thyroid autoimmunity.

Antigenic structure remains a major focus in thyroid immunology. The genes for three major thyroid antigens--thyroglobulin, thyroid peroxidase and the thyrotropin receptor--were sequenced in the late 1980's, and epitopes for antibody and T cells have been reported within the last year. In addition, new evidence for selective use of T-cell receptor V gene segments in human thyroid infiltrates may point the way to specific immunotherapy.

Identification of a thyroxine-containing self-epitope of thyroglobulin which triggers thyroid autoreactive T cells.

Although thyroglobulin (Tg), the thyroid prohormone, is well known as a T cell dependent autoantigen in human and experimental autoimmune thyroid disease, very little is known about the molecular basis of Tg recognition by T cells. In this paper, we have characterized the epitopes recognized by two clonotypically distinct, murine Tg autoreactive T cell hybridomas, CH9 and ADA2. In vitro iodination of a Tg preparation which was deficient in in vivo organified iodine was first used to confirm our previous observation that these T cells recognize iodination-related epitopes in the Tg molecule. Affinity chromatography of tryptic peptides derived from normally iodinated human Tg revealed that these epitopes were exclusively located in thyroxine (T4) containing peptides. Through the use of synthetic T4-containing peptides, representing the four major hormonogenic sites in Tg, we demonstrated that both CH9 and ADA2 recognize an epitope containing the T4 at position 2553 in human Tg. Sets of overlapping 5mer to 12mer peptides around this T4 showed that the most potent peptide was a 9mer beginning at Asp 2551. The T4 was shown to be a critical residue, since its replacement with any of the 20 naturally occurring amino acids produced only nonstimulatory peptides. Since the T cell hybridomas could also be stimulated by major histocompatibility complex class II positive (interferon-gamma-treated) thyroid epithelial cells in vitro, and their parent T cell lines can induce thyroiditis on adoptive transfer, the T4-containing Tg sequence described here is implicated as a pathogenic epitope in murine thyroid autoimmunity.

In vitro regulation of thyroglobulin (Tg) autoantibody production by Tg-specific T-cell lines and hybridomas.

To define the interactions between self thyroglobulin (Tg)-reactive T and B we co-cultured enriched B cells taken from rat or mouse Tg-primed mice with major histocompatibility complex (MHC) class II-restricted T-cell lines specific for iodinated determinants on self-Tg, or hybridomas derived from those lines. Using two clonally distinct T-cell hybridomas, ADA2 and CH9, in vitro help for Tg autoantibody responses was observed using mouse (M)Tg-primed B cells and a 100 ng/ml MTg challenge. Using rat Tg-primed B cells and the same conditions, only CH9 provided help, indicating that the fine specificity of B cells influences their ability to interact with specific anti-Tg T-cell clones. In contrast to T-cell hybridomas, their parent T-cell lines MTg9B3 and MTg12B suppressed Tg autoantibody responses in vitro, although they augmented bystander proliferation of unprimed B cells. The MTg12B cells also (i) diminished the survival of Tg-primed B cells, and (ii) inhibited the proliferation of an antigen-presenting B-cell hybridoma (LK35.2) in a cytostasis assay. These findings together support the view that their suppressive activity is mediated through cytotoxicity. While the role of class II-restricted cytotoxic cells in thyroid autoimmunity is unknown, the results suggest that such cells may act to suppress autoantibody responses as well as to mediate tissue damage to class II-expressing thyroid cells.

Class II-restricted bifunctional T-cell hybridomas reactive to self- and foreign myelin basic protein.

To study the cross-reactivity and functional properties oF murine T cells specific for myelin basic protein (MBP), a panel of 15 interleukin-2(IL-2)-releasing T-cell hybridomas was produced from SJL/J mice immunized either with human MBP or alternatively with a peptide corresponding to the known encephalitogenic sequence for SJL/J mice at positions 89-106. Hybridomas were I-As-restricted and activated by an MBP challenge as low as 20 nM. Cross-reactivity to other MBP indicated at least three immunodominant specificities for xenogeneic determinants, which could be further subdivided on the basis of antigen-independent reactivity to allogeneic stimulator cells. In addition, two self-specificities were demonstrated, one of which was to a determinant outside the 89-106 region. Irrespective of specificity pattern (self or foreign), all hybridomas effected antigen-dependent cytotoxicity of an antigen-presenting B-cell hybridoma (LS-102.9), which was mediated by cell contact or at close range. These findings suggest an approach to identifying new autoantigenic epitopes on MBP, and to studying T-cell-mediated effector pathways in myelin autoimmunity.

Cellular infiltration in induced rat thyroiditis: phenotypic analysis and relationship to genetic restriction.

We have investigated the responsiveness to thyroglobulin (Tg) plus complete Freund's adjuvant (CFA) and B. pertussis in a variety of inbred and MHC congenic strains of rats in terms of both Tg-autoantibody titres and histological thyroiditis index. Severity of thyroiditis was strongly Tg-dependent and closely related to the RT.1-MHC haplotype. Phenotypic examination of the inflammatory thyroid infiltrate using single and double indirect immunofluorescence techniques revealed a high proportion of macrophages and T lymphocytes, mainly of the cytotoxic/suppressor subset, in the high responder strains. Thyroid epithelial class II MHC expression although not prominent was strain-restricted and related to the amount of Ia+ leukocyte infiltrate.

Epitope specificity of spontaneous and induced thyroglobulin autoantibodies in the rat.

We have investigated the epitope specificities of rat thyroglobulin (Tg) autoantibodies arising either spontaneously in BB hybrid and BB rats or following induction in normal rats with thyroglobulin and adjuvant. Using a panel of thyroglobulins from different animal species it was possible to identify three different patterns of reactivity. These were: 1) recognition of all species of thyroglobulin; (2) recognition restricted to rat and mouse thyroglobulins and 3) recognition biased towards dog, rat and mouse thyroglobulins. Furthermore, using human thyroglobulin manifesting different levels of iodination, it was possible to show that sera with recognition pattern 1 recognized the iodination site of thyroglobulin and that this was inhibitable by thyroxine. Taken together these data provide evidence of restricted epitope recognition by Tg autoantibodies in the rat.

Critical role of iodination for T cell recognition of thyroglobulin in experimental murine thyroid autoimmunity.

We have used two clonotypically distinct thyroglobulin (Tg)-specific, I-Ak restricted monoclonal T cell populations to investigate the role of thyroid peroxidase-catalyzed iodination in Tg recognition by autoreactive T cells. The results showed that these T cells could recognize Tg only it it was sufficiently iodinated. Unlike normal mouse Tg, noniodinated mouse Tg was unable to induce significant thyroid lesions but could trigger the production of Tg autoantibodies. In these experiments, the importance of T cell recognition of iodination-related epitopes was emphasized by the inability of serum antibodies to distinguish Tg on the basis of iodine content, whether they were induced with normal or noniodinated Tg. Therefore, thyroid peroxidase-dependent modification of Tg would appear to be central to its recognition by autoreactive T cells and hence its capacity to induce autoimmune thyroid lesions.

Cytotoxicity of tumor necrosis factor for thyroid epithelial cells and its regulation by interferon-gamma.

The FRTL-5 line of differentiated rat thyroid epithelial cells was shown to be sensitive to the cytotoxic action of recombinant tumor necrosis factor. The sensitivity of the cells varied with the conditions of culture. It was markedly increased by preincubation with recombinant interferon-gamma, and the magnitude of this enhancement was affected by the presence of thyroid-stimulating hormone. The increased susceptibility of the cells to the cytotoxicity of tumor necrosis factor was clear as early as 2-4 h after the addition of interferon-gamma and greatly preceded the induced expression of major histocompatibility complex class II antigen. Since cells secreting interferon-gamma and tumor necrosis factors may co-exist in inflammatory infiltrates, our observations suggest that these factors may be early mediators of cell destruction in autoimmune thyroid disease.

Recognition of thyroglobulin autoantigenic epitopes by murine T and B cells.

We have used a large panel of thyroglobulins (Tg) prepared from a wide range of mammalian species to study the Tg autoantigenic epitopes recognized by populations of monoclonal and polyclonal murine T and B cells. This approach showed the existence of at least six different epitopes; three recognized by T cells (in association with I-Ak on antigen-presenting cells) and three by B cells (monoclonal antibodies). The majority of serum and monoclonal autoantibodies were found to be highly specific for mouse Tg, with some cross-reactive binding to rat Tg. In contrast, T-cell lines/clones and hybridomas recognized cross-reactive epitopes on Tg that were highly conserved throughout most of the mammalian orders. Moreover, two hybrid clones, which showed similar patterns of cross-reactivity, differed in their responsiveness to tryptic digests of human Tg. Thus, autoreactive T and B cells recognize distinct areas of the Tg molecule.

Interferon-mediated enhancement of thyroid major histocompatibility complex antigen expression. A flow cytometric analysis.

Epithelial expression of class II antigens encoded by the major histocompatibility complex (MHC) has been proposed as a means by which autoimmune thyroid disease may be initiated and maintained. We studied a rat thyroid epithelial cell line (FRTL-5), which constitutively expresses class I (OX18) but not class II (OX6 or OX17) determinants to quantify in vitro MHC antigen induction using flow cytometry. Recombinant rat gamma interferon (rIFN-gamma) induced dose-dependent expression of OX6 (I-A) antigen at greater than 48 h (maximum 80-90% of cells in culture at 100 U/ml), which was abrogated by DB-1, a monoclonal antibody to rat IFN-gamma. OX17 antigen (I-E) was also induced (86%) and OX18 (class I) markedly increased under these conditions. Other thyroid-active agents including the calcium ionophore A23187, dibutyryl cyclic AMP, thyroid-stimulating autoantibodies from Graves' disease patients (LATS), and TSH, caused no I-A induction. Supernatants from spleen cells stimulated with plant lectins (concanavalin A or phytohaemagglutinin), but not lectin alone, evoked substantial class II induction, which was inhibited by DB-1. These findings suggest that IFN-gamma is the central mediator of thyroid epithelial class II expression. FRTL-5 provides a powerful model for the analysis of thyroid MHC class II dynamics and a potential means of analysing the role of epithelial class II in autoimmune pathogenesis.

High efficiency antigen presentation by thyroglobulin-primed murine splenic B cells.

B cells primed in vivo with mouse or rat thyroglobulin present these antigens at very low concentrations to CH9, an Ly 1+2- T cell hybridoma specific for mouse and rat thyroglobulin. Presentation measured by interleukin 2 release from CH9 is sensitive to treatment with a monoclonal antibody eliminating splenic B cells but is unaffected by anti-Thy-1.2 or 33D1 (which destroy T cells and dendritic cells, respectively). Presentation is specific for the priming antigen and is blocked by preincubation of the B cells with sheep anti-mouse F(ab')2. We suggest that in this system, primed B cells present thyroglobulin and that this may represent a means by which an initial triggering event priming both B and T cells could allow maintenance of autoreactive responses in vivo in the presence of low concentrations of circulating antigen.

T-cell hybridomas specific for self and foreign thyroglobulins.

We have used somatic cell fusion techniques to produce and characterize murine T-cell hybridomas with specificity for self and foreign thyroglobulin (Tg). Two interleukin-2 (IL-2)-releasing I-Ak-restricted hybrid clones with specificity for self determinants on syngeneic Tg were derived from Tg-specific T-cell lines. These two autoreactive hybridomas were independently derived and were clonotypically distinct as determined by restriction fragment length polymorphism of the Ti beta-chain gene, but showed a similar pattern of cross-reactivity against rat and human (but not porcine) Tg. A third T-cell hybridoma showed a previously unknown specificity for the immunizing (non-inbred) Tg, but not for syngeneic Tg, indicating responsiveness to an allelic determinant. Although T-cell hybridization techniques have previously had only minimal application in experimental autoimmunity, this represents an approach to the study of Tg-specific T-cell responses at the molecular level.