T cell receptor gamma/delta chain diversity.

The TCR-gamma and -delta chains of six murine hybridomas were compared by one-dimensional SDS-PAGE and two-dimensional NEPHGE/SDS-PAGE analysis. This allowed the identification of three distinct gamma chains (gamma a, gamma b, and gamma c) and three distinct delta chains (delta a, delta b, and delta c). Four gamma/delta chain combinations (gamma a delta a, gamma b delta b, gamma b delta c, and gamma c delta a) were observed. These results indicate that multiple forms of the delta chain are expressed and suggest that the delta chains are encoded for by an Ig-like rearranging gene. This delta chain polymorphism significantly enhances the potential diversity of TCR-gamma/delta, which may be of importance for a better understanding of the putative ligand(s) recognized by this receptor.

Monoclonal antibody labeling of mononuclear cell surface antigens in formaldehyde-fixed paraffin-embedded cutaneous tissue.

The influence of the sequential stages of conventional formaldehyde fixation and paraffin embedding of cutaneous tissue on monoclonal antibody labeling of cell surface antigens is described. The effects of variation in fixation time, dehydration, clearing, wax embedding, and enzyme treatment of cutaneous sections were examined. By curtailing fixation time, using cold ethanol dehydration, and limited cold clearing with xylene, immunoreactivity of several important monoclonal antibodies was retained. Wax embedding could be achieved at 58 degrees C for 1 h or by using low-melting-point wax at 42 degrees C for 3 h. Thus was derived an optimal processing procedure which afforded good tissue morphology and allowed reliable reproducible labeling by monoclonal antibodies to cell surface antigens.

In situ detection of class I and II major histocompatibility complex antigens in the rat central nervous system during experimental allergic encephalomyelitis. An immunohistochemical study.

To determine in situ localization of cells bearing major histocompatibility complex (MHC) class I or II antigens in the central nervous system (CNS), immunohistochemical examination was performed on CNS sections of Lewis rats sensitized for experimental allergic encephalomyelitis (EAE). Class I antigens identified by OX18 were detected on endothelial cells (EC) and cells with dendritic morphology (DC) of normal rats. OX18+ DC increased in number as the clinical signs of EAE became more severe, while the number of OX18+ EC in clinical EAE rats was not different from that of normal control rats. Infiltrating lymphocytes were always observed around OX18+ vessels. Double staining showed that OX18+ DC was negative for glial fibrillary acidic protein (GFAP). Cells with morphological features of oligodendroglia were not detected with OX18 in both normal control and EAE rats. MHC class II antigens (Ia antigens) were detected using three MAbs: OX3, OX6 and OX17. These three different MAbs essentially showed the same staining pattern. In normal controls, mononuclear cells in the subarachnoid space were stained positively, but no Ia+ parenchymal cells were detected. In EAE rats, Ia+ DC were first detectable in the white matter of the spinal cord at the preclinical stage, and increased in number as the disease progressed. On the other hand, double-staining with OX6 and anti-factor VIII-related antigen antiserum, or with OX3 and anti-vimentin antiserum demonstrated that endothelial cells even with lymphocyte cuffing were negative for Ia antigens. Based on the data obtained in the present study, the possible role of MHC class I and II antigens in the development of EAE is discussed.

Characterization of interstitial dendritic cells in human liver.

Sensitive immunofluorescence and immunoperoxidase techniques were used to test an extensive range of monoclonal antibodies for reactivity with Kupffer cells and interstitial dendritic cells (DCs) in cryostat-cut sections of human liver. Leucocytes with a dendritic cell morphology were identified with CD45 (antileucocyte common) reagents in portal tracts, predominantly around bile ducts, and these cells stained strongly for the HLA-DP, DQ, and DR antigens. Kupffer cells stained less intensely with anti-class-II reagents, particularly anti-HLA-DQ. The interstitial DCs expressed the LFA-1 antigen but failed to stain with CD11b, CD11c, and the defined T and B cell CD antibodies; nor did they stain with antibodies to FcR1, FcR11, FcRIII, or the C3b receptor. Of the myeloid monoclonal antibodies available from the 3rd Leucocyte Differentiation Antigen Workshop, only Y2/131, Ki-M7, Ki-M8, and a minority of CD14 antibodies stained DCs, whereas Kupffer cells showed a wider reactivity with antimacrophage antibodies including those of workshop groups 11, 15, 16, and other unique antibodies. A 2nd probable DC population was identified in the liver capsule that had a similar phenotype to portal interstitial DCs. Although some minor phenotypic differences between liver portal DCs and the phenotypes of Langerhans cells and isolated tonsil DCs were noted, our results support the view that there is a unique hemopoietic lineage of DCs. The presence of DCs, which stimulate strong allogeneic T cell responses, in the portal triads is consistent with the fact that the histologic changes of graft-versus-host disease seen in bone marrow transplantation and the lymphocytic infiltrate in a rejecting liver allograft occur predominantly in the periportal region.

Identification of antigen presenting cells in normal and transplanted human heart: importance of endothelial cells.

The nature of class II-positive cells in normal and transplanted human heart has been investigated using immunoperoxidase and dual-immunofluorescent techniques. In normal heart approximately 83% of DR expression can be accounted for by EN4+ endothelial cells, most of which express intercellular adhesion molecule 1 constitutively. Few cells bearing the leukocyte common antigen are found in normal heart; most of them are RFD7+ macrophages or T cells. There is a paucity of RFD1+ dendritic cells. In transplanted heart showing signs of rejection, the infiltrate consists of RFD7+, RFD1+, RFD7+, RFD1+ cells and T lymphocytes. The increased class II expression within these biopsies is confined to the infiltrating cells. Dual-immunofluorescence demonstrates that nearly all the RFD1+ cells are from the recipient. In conclusion, in normal heart presentation of allogeneic class II is by the intercellular adhesion molecule-1-positive endothelial cells. After transplantation, there is an influx of recipient cells of the macrophage/dendritic series which are probably able to process allogeneic class II.

Human peripheral blood accessory cell: isolation by hypotonic density gradient, functional, and phenotypical characterization.

In order to purify the human peripheral blood-derived accessory cell that cooperate with T lymphocytes in the process of mitogenic stimulation, we developed a new density gradient separation. This was based on the principle of hypotonic swelling of the cells to obtain a differential change of the buoyant densities of cells. By this method, we have obtained a highly accessory cell-depleted lymphocyte fraction whose proliferative response to sodium periodate stimulation was almost aborted. Another fraction containing high accessory cell activity was further divided into Fc-receptor-positive and -negative cells. The latter revealed the highest accessory activity for T lymphocyte periodate stimulation. The cells were characterized according to a number of markers and appeared to resemble lymphoid dendritic cells. Compared with the monocyte/macrophage fraction, they showed veils and dendritiform elongations and expressed reduced values of monocyte/macrophage specific markers. Compared with the high accessory activity of these cells, monocytes/macrophages expressed a low accessory activity.

Dendritic/Langerhans cells and prognosis in patients with papillary thyroid carcinomas. Immunocytochemical study of 106 thyroid neoplasms correlated to follow-up data.

Paraffin sections of 106 primary thyroid carcinomas were the subject of an immunocytochemical study to determine the density of infiltrates of S-100 protein-positive dendritic/Langerhans cells (LC), lysozyme-positive histiocytes, and LCA-positive lymphocytes. Evidence of dense infiltrates of LCs was found only in the majority of papillary thyroid carcinomas (PCs). The determination of the quantity of LCs proved to be a highly effective means of assessing the prognosis of these tumors. Irrespective of other morphologic and clinical features, no single instance of death resulting from cancer occurred among 23 PCs with dense LC infiltrates (including 6 tumors of stage pT4), while 9 of 53 (17%) of the remaining patients ultimately died from thyroid cancer. On the other hand, the degree of histiocytic and lymphocytic infiltrations was not associated with a distinct biologic behavior neither among PC nor among the remaining thyroid carcinomas. These findings suggest that LCs may play an important role in the immunologic defense mechanisms of the host against the tumor only in the papillary type of thyroid cancer.

Significance of S-100 protein immunostaining in the immunohistological analysis of normal and neoplastic lymphoid tissues--an appraisal.

S-100 protein is a heterogeneous fraction of dimeric polypeptides (alpha and beta subunits) that can exist in different combination forms within the various tissues. Concerning the S-100 protein immunodetection within lymphoid tissue, the heterogeneity of the S-100 antigen, the tissue quality (frozen or paraffin-embedded after treatment with different fixatives) and the treatment of the tissue with different immunostaining methods and antibodies of different nature, all make for inconsistent results obtained in the immunohistological studies reported in the literature. Most of the S-100-positive cells of the lymphoreticular system are dendritic cells involved in the immune response (interdigitating reticulum cells, Langerhans cells, and follicular dendritic reticulum cells), other S-100-positive cells belonging to the mononuclear/phagocytic system. S-100 protein immunostaining may be used as a helpful immunohistological diagnostic clue to certain malignancies of the immune system (follicular center cell lymphomas) on the basis of their specifically related dendritic cell microenvironment. In addition to monoclonal antibodies for the immunophenotypic characterization of dendritic cells and macrophages and to enzyme reactions, the combined use of anti-S-100 antibodies specific for each of the S-100 protein subunits, tested with sensitive procedures, would be a very useful tool in the attempt to classify the proliferative disorders of dendritic cells and macrophages.

Dendritic cells in bronchial adenocarcinoma.

We studied 28 bronchial adenocarcinomas (including 11 bronchiolo-alveolar carcinomas) stained for S-100 protein by the PAP technique in order to visualize dendritic cells in the neoplastic area. Dendritic cells were found in all cases varying in number from single to multiple and from region to region. Bronchiolo-alveolar carcinomas contained a similar quantity of these cells. The most numerous dendritic cells up to several scores per microscopical low-power field almost in all fragments of the tumour were found in three adenocarcinomas with numerous foci of the squamous cell metaplasia. Normal and metaplastic squamous epithelium of the bronchial mucosa did not show the presence of dendritic cells.

A 93-kDa glycoprotein expressed on human cultured monocytes and dendritic reticulum cells defined by an anti-K562 monoclonal antibody.

An IgG2a monoclonal antibody, referred to as 12B1 and raised against the K562 cell line, reacted with adherent monocytes maintained in culture for several days but not with bone marrow or peripheral blood cells including freshly isolated monocytes. Among human leukemic cell lines, 12B1 reacted essentially with the promyelocytic HL60 cell line. 12-O-Tetradecanoylphorbol 13-acetate treatment, but no other differentiation inducer, strongly enhanced its reactivity on K562, HL60 and the histiocytic U937 cell line. Immunoperoxidase staining of sections of normal human tissues showed that 12B1 specifically recognized dendritic reticulum cells in germinal centers of lymph nodes, spleen and tonsils. The 12B1-detected antigen is a highly glycosylated polypeptide of an apparent molecular mass of 93-86 kDa. The 12B1 antigen appears to be a new glycoprotein marker shared by adherent monocytes and dendritic reticulum cells. The association of the 12B1 epitope with cells which present antigen and/or exert accessory function suggests that this molecule could play a role in these activities.

S-100 protein-positive dendritic cells in colorectal adenocarcinomas. Distribution and relation to the clinical prognosis.

Dendritic cells (DC) in 121 colorectal adenocarcinomas were investigated immunohistochemically, using anti-S-100 protein antibody. S-100(+)DC were recognized among the malignant cells and/or around the tumor and differed in distribution either from lysozyme-positive macrophages or from neuron-specific enolase-positive neural tissue. Patients with many S-100(+)DC (more than 30 cells per 10 high-power fields) in the tumor survived longer than did those with few such cells (less than 30 cells), most often with no metastases (P less than 0.001). The grade of S-100(+)DC infiltration was related to both density of lymphocytic infiltration in the primary tumor and the degree of paracortical hyperplasia in the regional lymph nodes (P less than 0.05). Dendritic cells, therefore, as antigen-presenting cells, conceivably mediate cell immunity in a tumor with lymphoid infiltration and in the regional lymph nodes. The number of S-100(+) DC in the primary colorectal carcinomas represents one aspect of such a series of antitumor immunoreaction, in vivo.

The cell surface of mouse dendritic cells: FACS analyses of dendritic cells from different tissues including thymus.

The surface of dendritic cells from mouse spleen, thymus, and epidermis has been compared with a panel of monoclonal antibodies and the FACS. A method was first developed to isolate populations of large, adherent, thymic dendritic cells that were greater than 90% pure. These were released by collagenase digestion and separated from adherent macrophages after overnight culture. Enrichment was based on the facts that most macrophages remained plastic adherent and rosetted strongly with antibody-coated erythrocytes. As in spleen, thymic dendritic cells were stellate in shape, had abundant class I and II MHC products, lacked many standard macrophage and lymphocyte markers, and actively stimulated the mixed leukocyte reaction. Most spleen and thymic dendritic cells could be lysed by the 7D4 mAb, to the low-affinity IL-2 receptor, and complement but the levels of 7D4 by FACS were low and sometimes not above background. Differences among dendritic cells from different tissues were noted with other mAb. Adherent dendritic cells from thymus all expressed the J11d "B cell" antigen and the NL145 interdigitating cell marker, but lacked the 33D1 spleen dendritic cell antigen. Eighty to ninety percent of spleen dendritic cells were J11d-, NL145-, 33D1+ but the remainder expressed the J11d+, NL145+, 33D1- thymic phenotype. The latter phenotype also was identical to that of epidermal Langerhans' cells. We postulate that the major 33D1+ cell in spleen represents a migratory stage in which dendritic cells are moving from tissues to lymphoid organs.

Properties of lymph-borne (veiled) dendritic cells in culture. II. Expression of the IL-2 receptor: role of GM-CSF.

Fresh lymph-borne (veiled) dendritic cells (L-DC) in the rat are almost totally negative for the interleukin-2 (IL-2) receptor detected by the monoclonal antibody (mAb) MRC OX39. After 16 hr culture more than 90% of L-DC are OX39 positive, and increased levels of expression can be seen within 5 hr culture. In cultures of L-DC and allogeneic lymphocytes. L-DC appear to express the IL-2 receptor more rapidly than lymphocytes. The intensity of labelling of L-DC is variable but maximal levels are similar to those seen on lymphoblasts. Culture in the presence of concanavalin A (Con A)-stimulated spleen cell supernatants or recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) results in a more rapid and intense expression of the IL-2 receptor by L-DC. L-DC cultured following rigorous T-cell depletion, or derived from athymic rats also express the IL-2 receptor after culture with GM-CSF. Cultured, but not fresh, L-DC bind iodinated recombinant IL-2 in a dose-dependent manner and binding is inhibited by excess unlabelled ligand. The amount of IL-2 bound varies but maximal amounts are similar to those bound by lymphoblasts. Following intravenous endotoxin injection, a large proportion of freshly collected L-DC express the IL-2 receptor and the number of L-DC released into the lymph is increased. An antibody to the IL-2 receptor which blocks an allogeneic MLR has no effect on a xenogeneic MLR using rat L-DC as stimulators and mouse lymphocytes as responders.

Immunohistochemical study of bone marrow sections in CLL.

T lymphocytes and dendritic reticulum cells (DRC) were studied in frozen-cut bone marrow sections of 35 patients with chronic lymphocytic leukemia (CLL) (infiltration patterns: interstitial 8, nodular 6, mixed 9, diffuse 12) and 13 cases of low grade non Hodgkin's lymphoma (NHL) (centroblastic/centrocytic 7, centrocytic 3, lymphoplasmacytoid 3) with bone marrow involvement. In contrast to the usual findings in normal bone marrow, in CLL and low grade NHL CD4 positive cells were more numerous than CD8 positive cells. Whereas in NHL CDR were large and occupied all the nodule, in CLL were small and located in the center of the nodule. These findings can be of interest in the study and differential diagnosis of lymphoproliferative disorders.

Identification and characterization of a human cell line with dendritic cell features.

CORP-4 is a cell line obtained in our laboratory from an explanted human bladder carcinoma. This cell line shows certain dendritic cell features such as adherence to the culture plate surface, a doubling time of 24 h and an enzymatic profile typical of cells involved in antigen presentation (non-specific esterases, lysozyme and alpha-1-antitrypsin). Its phenotypic analysis revealed CD 15 and Fc receptor expression, S-100 surface protein and the presence of positive reactivity to different lectins such as Concanavalin A (Con A) and Peanut agglutinin (PNA). CORP-4 was found to be a non-phagocytic cell line after it was assayed with latex, and FcR- and C3bR-mediated phagocytosis. Furthermore, CORP-4 produced interleukin-1 (IL-1) as determined by thymocyte proliferation assays and also fixes immune complexes in a non-complement dependent fashion. HLA class I and class II antigens were inducible by both 5 azacytidine and gamma interferon.

Delta-chains of dendritic epidermal T cell receptors are diverse but pair with gamma-chains in a restricted manner.

To assess the diversity of TCR delta-gene expression in dendritic epidermal T cells (DETC), we characterized the delta-gene used by a panel of cell lines that express the gamma delta TCR on the cell surface. Northern hybridization analyses with a panel of V delta probes representing each of the six known V delta families showed that each of these lines expressed either V delta 1 or V delta 6 containing transcripts. Southern hybridization analysis with the V delta probes gave rearrangement patterns consistent with Northern hybridization results. The correlation of V delta expression with V gamma expression revealed that not only may V delta usage be restricted in DETC cells, but that the pairing of gamma- and delta-chains may not be random. In the DETC cells examined, V delta 1 chains paired exclusively with V gamma 3C gamma 1 chains and V delta 6 chains paired with C gamma 2 and C gamma 4 chains. Sequence analysis of delta-cDNA clones corresponding to the expressed delta-chains from one of the V delta 1 expressing cell lines and two of the V delta 6 expressing cell lines revealed that, although the V delta 1 gene segment sequence and one of the V delta 6 gene segment sequences were identical to previously published V delta sequences, considerable variable region diversity was generated by complex V-D-J rearrangement patterns that utilized various combinations of N-additions, D delta 2 and possibly D delta 1 segments, and J delta 1 or J delta 2 segments. These complex rearrangement patterns distinguish these DETC lines from early thymocytes and suggest that, if DETC are thymic derived, they originate from relatively mature thymic cells.

Tonsillar epithelial dendritic cells. Demonstration by lectin binding, immunohistochemical characterization, and ultrastructure.

The association of tonsillar epithelium with lymphoid tissue suggests a functional relationship in the generation of local immune responses analogous to skin. Therefore, we investigated this epithelium for the presence of dendritic cells similar to epidermal Langerhans cells. Clusters of tonsillar epithelial dendritic cells (TEDC) were revealed by binding of the lectin peanut agglutinin, a feature not previously directly demonstrated for this cell type. Similar to epidermal Langerhans cells, TEDC showed immunoreactivity for antibodies to S-100, T6, and Ia antigens. Ultrastructurally, TEDC had features most similar to epidermal indeterminate cells. In addition, the tonsillar epithelium showed regional variation in the types of intraepithelial lymphocytes present. Surface epithelium contained T cells predominantly of helper/inducer phenotype, while the crypt epithelium contained a mixture of B cells, plasma cells, and T cells of both helper/inducer and suppressor/cytotoxic phenotypes. These findings, together with the demonstration of TEDC, suggest that the tonsillar epithelial microenvironment, like skin, provides a site of cell-cell interactions important in initiating immune responses.

Selective visualization of human dendritic reticulum cells in reactive lymphoid follicles by the immunohistochemical demonstration of the subunit A of factor XIII (F-XIIIa).

Immunohistochemical techniques have been used to localize clotting factor XIII subunit A in human reactive lymphoid follicles. The follicular dendritic reticulum cells (DRCs) were identified by the monoclonal antibodies R4/23 and OKB-7 as well as by their 5'-nucleotidase positivity. Follicular histiocytic reticulum cells (HRCs) were demonstrated by their acid phosphatase and non-specific esterase reactions. Capillaries were selectively visualized by adenosine triphosphatase. The immunohistochemical demonstration of F-XIIIa was preferably carried out in combination with one or two of the above marker techniques, on the same cryostat section. The subunit A of factor XIII is present in follicular DRCs. Their selective immunohistochemical demonstration with antibody against F-XIIIa requires formaldehyde fixation of cryostat sections. Similar fixation, however, is inappropriate for the demonstration of F-XIIIa reactivity of DRCs in paraffin sections. For this purpose, acetic acid-formalin fixation is useful. Follicular HRCs are consistently negative for F-XIIIa, contrary to the F-XIIIa positivity of sinusoidal and interfollicular HRCs. Developmental and functional implications of F-XIIIa reactivity in DRCs and HRCs are suggested.

Phenotypic expression of cells of stationary elements in human lymphoid tissues. A histochemical and immunohistochemical study.

The stationary elements of lymphoid tissues are composed of four types of cells: histiocytes, interdigitating reticulum cells, follicular dendritic cells, and fibroblastic reticulum cells. The phenotypes of these cells were determined with a large panel of monocyte/histiocyte, C3 receptor monoclonal antibodies, and others. Based on the monocyte-marker expression, histiocytes can be separated into two groups: (1) free histiocytes (monocyte-marker positive) and (2) fixed histiocytes (monocyte-marker negative). The former are characterized by the expression of monocyte markers, such as OK M1, Co Mo2, BRL Mol/Mo2, and Leu M3, whereas the latter are not. Interdigitating reticulum cells are localized in the T-cell zone. These cells are characterized by the expression of 1E9, 2H9, and Leu M1. Interdigitating reticulum cells and fixed histiocytes are similar in terms of marker expression and enzyme histochemistry. However, in interdigitating reticulum cells, the Leu M1 antigen is localized on the membrane, in contrast to histiocytes, in which it has a Golgi distribution. Follicular dendritic cells are present in germinal centers and mantle zones. These cells express complement (C3) receptors and several monocyte markers (including OK M1 and Co Mo2). Follicular dendritic cells are capable of trapping antigens onto their membranes. This unique property makes us reluctant to conclude that follicular dendritic cells are related to monocytes. Fibroblastic reticulum cells express BA-1 and alkaline phosphatase, and they form a dendritic network, especially in the T-cell zone. The results of this study demonstrate that immunoperoxidase staining with monoclonal antibodies can reveal the distribution of histiocytes and dendritic/reticulum cells in lymphoid tissues.