Immunohistochemical analysis of mycosis fungoides on paraffin-embedded tissue sections

Mycosis fungoides (MF) is typically characterized by dermal and epidermal infiltration of T lymphocytes with a helper/inducer phenotype. Immunophenotypic analysis of such cases was traditionally performed by flow cytometry or immunohistochemistry on cryostat sections. With the advent of new monoclonal antibodies developed against T-cell antigens, including CD3, CD4, CD5, and CD8, it is now possible to immunophenotype T-cell subpopulations in paraffin-embedded tissues. To investigate the potential use of these antibodies for the evaluation of cutaneous lesions, 35 specimens (34 skin and 1 lymph node) from 29 patients with MF were retrospectively reviewed and immunophenotyped in paraffin sections with antibodies to CD3 (T-cell CD3), CD4 (NCL-CD4-1F6), CD5 (NCL-CD5-4C7), CD8 (CD8/144B), and CD20 (L26). Epidermal and dermal distribution of T and B cells were analyzed, and we assessed the ratios of CD4+ to CD8+ T cells. All of our 35 cases demonstrated a predominant CD3+ T-cell population. In 32 cases, the neoplastic cells expressed CD3, CD4, and CD5 consistent with a T-helper/inducer phenotype. In three cutaneous cases, the neoplastic CD4+ T cells showed minimal or absent expression of CD5, indicating an aberrant phenotype. In the majority of cases, minimal CD8+ T cells were present in the background, but in four cases, the CD4:CD8 ratios were 2:1 or less. Thirty-two cutaneous cases demonstrated epidermotropism exclusively by CD4+ T cells; one case showed both CD4+ and CD8+ T cells. In 17 cutaneous cases, scattered dermal CD20+ B cells were found individually or in small clusters within the background surrounding the neoplastic infiltrates. We concluded, therefore, that the immunophenotypic analysis of T-cell subpopulations using monoclonal antibodies of CD3, CD4, CD5, and CD8 was useful for histologic evaluation and confirmation of MF lesions in paraffin-embedded tissue. These antibodies might also provide an effective method of immunophenotyping other neoplastic and non-neoplastic T-cell populations in paraffin-embedded tissues.     

Active immunity against the CD4 receptor by using an antibody antigenized with residues 41-55 of the first extracellular domain

Using the process of "antibody antigenization," we engineered two antibody molecules carrying in the third complementarity-determining region of the heavy chain variable domain a 7-mer or a 15-mer peptide epitope of the first extracellular domain (D1) of human CD4 receptor--namely, Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser (SFLTKGPS; positions 42 through 49) and Gly-Ser-Phe-Leu-Thr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-Arg-Ala (GSFLTKGPSKLNDRA; positions 41 through 55). These amino acid sequences are contained in the consensus binding site for the human immunodeficiency virus (HIV) on CD4 receptor. Both antigenized antibodies (AgAbs) bound recombinant gp120 and were recognized by a prototype monoclonal antibody to CD4 whose binding site is within amino acid residues 41-55. AgAbs were then used as immunogens in rabbits and mice to elicit a humoral response against CD4. Only the AgAb carrying the sequence 41GSFLTKGPSKLN-DRA55 induced a response against CD4. The induced antibodies showed specificity for the amino acid sequence of CD4 engineered in the AgAb molecule, were able to inhibit the formation of syncytia between human CD4+ T cells MOLT-3 and 8E5 (T cells that are constitutively infected with HIV), and stained human CD4+ CEM T cells. Four murine monoclonal antibodies were used to analyze the relationship between syncytia inhibition and CD4 binding at the single antibody level, and indicated that recognition of native CD4 is not an absolute requirement for inhibition of syncytia. This study demonstrates that antigenized antibodies can be used as immunogens to elicit site-specific and biologically active immunity to CD4. The importance of this approach as a general way to induce anti-receptor immunity and as a possible new measure to immunointervention in HIV infection is discussed.     

Monoclonal anti-CD44 antibody acts in synergy with anti-CD2 but inhibits anti-CD3 or T cell receptor-mediated signaling in murine T cell hybridomas

We investigated the effects of anti-murine CD44 monoclonal antibodies on the activation of antigen-specific T cell hybridomas. Anti-murine CD44 antibodies by themselves did not induce the production of IL-2 by antigen-specific T cell hybridomas. However, anti-murine CD44 monoclonal antibodies were able to either inhibit or enhance the production of IL-2, depending on the other monoclonal antibodies used as comitogenic stimuli. When a T cell hybridoma was activated by antigen and antigen-presenting cells or anti-CD3 antibodies, addition of anti-CD44 antibodies inhibited IL-2 production. In contrast, monoclonal anti-CD44 antibodies acted in synergy with anti-human CD2 antibodies in stimulating a murine T cell hybridoma stably transfected with the human CD2 gene to produce IL-2. Therefore, cross-linking of surface CD44 is able to deliver either a positive or a negative signal in murine antigen-specific T cell hybridomas. One of the ligands for CD44 is hyaluronic acid. Hyaluronic acid in vitro significantly increased the activation of murine T cell hybridomas. Hyaluronic acid itself was mitogenic for T cell hybridomas. Therefore, in addition to being an adhesion molecule, CD44 functions as a signal-transducing molecule on murine T cell hybridomas.     

A B7.1-antibody fusion protein retains antibody specificity and ability to activate via the T cell costimulatory pathway

We describe the construction and characterization of an Ab fusion protein specific for the tumor-associated Ag HER2/neu linked to sequences encoding the extracellular domain of the B7.1 T cell costimulatory ligand. The Ab domain of the fusion molecule will specifically target HER2/neu-expressing tumor cells, while the B7.1 domain is designed to activate a specific immune response. We show that the B7.1 fusion Ab retained ability to selectively bind to the HER2/neu Ag and to the CTLA4/CD28 counter-receptors for B7.1. Specific T cell activation was observed when the B7.1 Ab fusion protein was bound to HER2/neu-expressing cells. The use of the B7.1 Ab fusion protein may overcome limitations of gene transfer and/or standard Ab therapy and represents a novel approach to the eradication of minimal residual disease.     

T-cell infiltration of primary CNS lymphoma

We stained 13 primary CNS lymphomas (PCNSLs) (six from patients with AIDS, seven from immunocompetent patients) with a panel of antibodies to T cells (pan T cell [CD3], T helper cell [CD4], T suppressor cell [CD8], delta/delta cell [CD4-8-]), B cells (CD20), hematopoietic cells (T200), and NK cell (CD56). We estimated the percentage of tumor cells staining with each antibody. All tumors were B-cell lymphomas. The non-AIDS tumors showed a significant infiltration with CD3+ cells (mean of 10.82% of total cells). The AIDS patients' tumors showed a smaller percentage of CD3+ infiltrating cells (mean, 4.88% of total cells) (p<0.01). CD4+ cells were 9.11% of the total hematopoietic cells in the non-AIDS patients and 3.13% in AIDS patients (p<0.01). AIDS patients showed some CD8+ cells (0.3%), which was significantly higher than in immunocompetent patients (0%) (p<0.05). Very few tumor cells stained with the NK cell and delta/delta cell markers. Both immunocompetent and AIDS patients with PCNSL exhibit significant CD3+ and CD4+ cell infiltration of their tumors; this infiltration is significantly lower in AIDS patients. AIDS patients show a minor CD8+ cell infiltration of their tumors. These results on PCNSL are different from systemic lymphomas, which show a higher CD4 and CD8 cell infiltration, and may offer insights into the more aggressive nature of AIDS-related PCNSL.     

Immunoreactivity of a new CD5 antibody with normal epithelium and malignant tumors including thymic carcinoma

CD5, first recognized on subsets of lymphocytes, also is detected in thymic carcinoma but not in thymoma or other malignant tumors. We studied CD5 expression in 73 cases of malignant tumors of various organs, 22 cases of thymoma, and 7 cases of thymic carcinoma by immunohistochemistry using the new monoclonal anti-CD5 antibody, NCL-CD5-4C7, with a pressure cooker antigen retrieval method. All cases of thymic carcinoma showed positive staining for CD5, predominantly on the cell membrane. Two of 4 cases of atypical thymoma also showed focal positivity, whereas the other types of thymoma were negative. CD5 was detected in cases of malignant tumors other than squamous cell carcinoma and in the normal epithelium of their counterparts. Squamous cell carcinomas of various organs were negative for CD5. Malignant mesothelioma showed peculiar intracytoplasmic staining in contrast to the other tumors. The NCL-CD5-4C7 positivity in thymic epithelial tumors may support the hypothesis suggesting progression of atypical thymoma to thymic carcinoma. NCL-CD5-4C7 may be useful in the differential diagnosis of mediastinal tumors, especially between thymic carcinoma and metastatic squamous cell carcinoma of various primary sites, and for distinguishing malignant mesothelioma from adenocarcinoma of the lung by the different staining pattern.     

Naturally processed class II epitope from the tumor antigen MUC1 primes human CD4+ T cells

Epithelial cell mucin MUC1 is expressed on adenocarcinomas in an underglycosylated form that serves as a tumor antigen in breast, pancreatic, ovarian, and other tumors. Two predominant MUC1-specific immune responses are found in patients: CD8+ CTLs, which recognize tandemly repeated epitopes on the MUC1 protein core, and IgM antibodies. There have been no reports to date of MUC1-specific CD4+ T-helper cells in cancer patients. We show here that MUC1-specific CD4+ T cells are neither deleted nor tolerized and that CD4+ T cell responses can be generated when an appropriate soluble form of MUC1 is used. Naive CD4+ T cells from healthy donors were primed in vitro to a synthetic MUC1 peptide of 100 amino acids, representing five unglycosylated tandem repeats, presented by dendritic cells. They produced IFN-gamma and had moderate cytolytic activity. We identified one core peptide sequence, PGSTAPPAHGVT, that elicits this response when it is presented by HLA-DR3.     

A modified cell surface marker gene for transgenic animal studies

We developed a marker gene encoding a human cell surface molecule called CD8 for use in transgenic animal studies. The CD8 cDNA contains three mutations: one in the extracellular domain which prevents interaction with its ligand MHC class I and the other two in the cytoplasmic domain which inhibit its signalling function. The cDNA was linked to a fragment of the human growth hormone gene and in transgenic animal studies, expression was observed in the appropriate cell types using a CD2 enhancer. The advantage of the CD8 marker gene is that it is incapable of signalling via its only known signalling pathway and its expression can be monitored using monoclonal antibodies and microscopy or flow cytometry.     

Interleukin 15 is produced by endothelial cells and increases the transendothelial migration of T cells In vitro and in the SCID mouse-human rheumatoid arthritis model In vivo

The capacity of endothelial cells (EC) to produce IL-15 and the capacity of IL-15 to influence transendothelial migration of T cells was examined. Human umbilical vein endothelial cells expressed both IL-15 mRNA and protein. Moreover, endothelial-derived IL-15 enhanced transendothelial migration of T cells as evidenced by the inhibition of this process by blocking monoclonal antibodies to IL-15. IL-15 enhanced transendothelial migration of T cells by activating the binding capacity of the integrin adhesion molecule LFA-1 (CD11a/CD18) and also increased T cell motility. In addition, IL-15 induced expression of the early activation molecule CD69. The importance of IL-15 in regulating migration of T cells in vivo was documented by its capacity to enhance accumulation of adoptively transferred human T cells in rheumatoid arthritis synovial tissue engrafted into immune deficient SCID mice. These results demonstrate that EC produce IL-15 and imply that endothelial IL-15 plays a critical role in stimulation of T cells to extravasate into inflammatory tissue.     

Redirecting effector T cells through their IL-2 receptors

Fusion proteins constructed of a tumor-specific Ab joined to IL-2 (Ab-IL-2) have been used in the past to deliver cytokine directly to the site of tumor cells in vivo. These molecules mimic the activity of IL-2 and assist in activating and expanding antitumor effector cells. To enhance the cytolytic activity of CTL specific for peptide epitopes of the Her-2/neu tumor Ag presented by HLA-A*0201 molecules, a fusion protein was constructed consisting of a single chain Ab specific for Her-2/neu, linked to IL-2 (neu-Ab-IL-2). When added to a mixture of tumor cells and Her-2/neu-specific CTL, the protein was found to augment lysis of tumor cells. In addition, the hybrid molecule also promoted lysis of Her-2/neu expressing tumors by non-tumor-specific cloned T cell lines, including Th1 CD4 cells. Analysis of the mechanism of cytotoxicity revealed that the fusion protein mediates the formation of stable conjugates between T cells expressing IL-2R and tumor cells expressing Her-2/neu, resulting in lysis through the Fas-Fas ligand pathway. Lysis induction was independent of specific engagement by the TCR. When tested for its ability to enhance tumor cell eradication by Her-2/neu-specific CD8+ T cells in an adoptive transfer model in SCID mice, neu-Ab-IL-2 facilitated the elimination of tumor cells in vivo. Surprisingly, the combination of non-tumor-specific CD8+ T cells and fusion protein also induced a significant delay of tumor growth. This represents a novel approach for redirecting non-tumor-specific T cells to eliminate tumors.     

Molecular genetic analysis of the pullulanase B gene of Bacillus acidopullulyticus

In this study we describe immunotoxins prepared with different CD2 monoclonal antibodies (mAbs) and a ribosome-inactivating protein, saporin. The CD2 immunotoxins were tested on different models. Anti-CD2-saporin conjugates inhibited protein synthesis by a neoplastic CD2+ cell line (SKW-3) and by an interleukin 2 dependent polyclonal CD2+ lymphoid cell culture (T lymphoblasts), with IC50s ranging from 10(-13) M to 10(-11) M (as saporin). Similar results were obtained with proliferation inhibition tests (3H-thymidine incorporation) on phytohaemagglutinin (PHA) driven lymphoid cultures and on mixed lymphocyte culture activated lymphocytes. Moreover a CD2-ricin A chain conjugate was less effective than an analogous immunotoxin containing the same CD2 mAb and saporin in inhibiting lymphocyte proliferation induced by PHA (IC50 approximately 10(-9) M as ricin A chain versus 10(-12) M as saporin). The conjugates were not toxic on bone marrow stem cells. These results suggest that CD2-saporin immunotoxins could represent an effective tool for CD2+ lymphomas or leukaemias, and for T-dependent immune disorders, such as transplanted organ rejection and graft-versus-host disease.     

Peptide-antibody conjugates for tumour therapy: a MHC-class-II-restricted tetanus toxin peptide coupled to an anti-Ig light chain antibody can induce cytotoxic lysis of a human B-cell lymphoma by specific CD4 T cells

Anti-idiotype antibody therapy of B-cell lymphomas, despite numerous promising experimental and clinical studies, has so far met with limited success. Tailor-made monoclonal anti-idiotype antibodies have been injected into a large series of lymphoma patients, with a few impressive complete tumour remissions but a large majority of negative responses. The results presented here suggest that, by coupling to antilymphoma idiotype antibodies a few molecules of the tetanus toxin universal epitope peptide P2 (830-843), one could markedly increase the efficiency of this therapy. We show that after 2-hr incubation with conjugates consisting of the tetanus toxin peptide P2 coupled by an S-S bridge to monoclonal antibodies directed to the lambda light chain of human immunoglobulin, human B-lymphoma cells can be specifically lysed by a CD4 T-lymphocyte clone specific for the P2 peptide. Antibody without peptide did not induce B-cell killing by the CD4 T-lymphocyte clone. The free cysteine-peptide was also able to induce lysis of the B-lymphoma target by the T-lymphocyte clone, but at a molar concentration 500 to 1000 times higher than that of the coupled peptide. Proliferation assays confirmed that the antibody-peptide conjugate was antigenically active at a much lower concentration than the free peptide. They also showed that antibody-peptide conjugates required an intact processing function of the B cell for peptide presentation, which could be selectively inhibited by leupeptin and chloroquine.(ABSTRACT TRUNCATED AT 250 WORDS)     

In situ characterization of gingival mononuclear cells in rapidly progressive periodontitis

Rapidly progressive periodontitis (RPP) has been suggested as a distinct clinical entity within the spectrum of early onset periodontitis. Immunological mechanisms have been considered in the pathogenesis of RPP. This study was designed to evaluate the distribution and phenotypic properties of the lymphocyte populations within the affected gingival tissue of patients with RPP. Biopsies were obtained from 16 patients between 22 and 33 years of age. The tissue samples were processed for both histopathological and immunohistochemical examinations. Gingival tissue T lymphocytes (CD3+), helper T cells (CD4+), suppressor-cytotoxic T cells (CD8+), and cells positive for HLA-DR antigen were identified using monoclonal antibodies with an immunoperoxidase technique. Intracytoplasmic immunoglobulin-containing cells were also stained immunohistochemically with polyclonal antibodies. CD3+ cells were mainly located beneath the pocket epithelium. CD4+ and CD8+ cells were evenly distributed within this T-cell infiltrate with a CD4+/CD8+ ratio of 1:12. Numerous HLA-DR+ cells were also observed in the lymphocytic infiltrates. The majority of mononuclear cells located throughout the stroma were IgG+ plasma cells. Our results indicate that RPP patients present an IgG-bearing plasma cell dominated lesion with equal participation of both T-cell subpopulations. These findings suggest that activation and proliferation of B-cells play an important role in the pathogenesis of periodontal diseases.     

Characterization of a Helicobacter pylori neutrophil-activating protein

Antibody-blocking studies have demonstrated the role of CD6 in thymocyte-thymic epithelial (TE) cell adhesion. Here we report that CD6 expressed by COS cells mediates adhesion to TE cells and that this interaction is specifically blocked with an anti-CD6 monoclonal antibody (mAb) or with a mAb (J4-81) that recognized a TE cell antigen. We isolated and expressed a cDNA clone encoding this antigen and show that COS cells transfected with this cDNA bind a CD6 immunoglobulin fusion protein (CD6-Rg). This antigen, which we named ALCAM (activated leukocyte-cell adhesion molecule) because of its expression on activated leukocytes, appears to be the human homologue of the chicken neural adhesion molecule BEN/SC-1/DM-GRASP. The gene was mapped to human chromosome 3q13.1-q13.2 by fluorescence in situ hybridization of cDNA probes to metaphase chromosomes. We prepared an ALCAM-Rg fusion protein and showed that it binds to COS cell transfectants expressing CD6, demonstrating that ALCAM is a CD6 ligand. The observations that ALCAM is also expressed by activated leukocytes and that both ALCAM and CD6 are expressed in the brain suggest that ALCAM-CD6 interactions may play a role in the binding of T and B cells to activated leukocytes, as well as in interactions between cells of the nervous system.     

Synthetic peptides derived from the fourth domain of CD4 antagonize off function and inhibit T cell activation

We have developed synthetic peptide analogs to analyze novel surface structures of the human CD4 protein potentially involved in T cell activation. Linear and cyclic peptides derived from the FG and CC' loops of the membrane proximal fourth domain of CD4 displayed inhibitory activities in a CD4-dependent immunological assay. These results suggest that the fourth domain of CD4 plays an important role in T cell activation. In addition, we report the synthesis of a highly stable CD4 peptide analog cyclized by the formation of an amide bond between amino and carboxyl termini. Serum stability studies showed that this main-chain cyclic CD4 peptide was highly resistant to proteolytic degradation while the linear and disulfide cyclic peptides were much less stable. The strategy of main chain cyclization of CD4 peptides may represent a promising approach to generate proteolytically stable, orally active immunoregulatory agents.     

MHC class I-restricted lysis of human oligodendrocytes by myelin basic protein peptide-specific CD8 T lymphocytes

Multiple sclerosis (MS) is considered to be an autoimmune disease that is directed either at myelin or at its cell of origin, the oligodendrocytes (OL). The inflammatory lesions in the central nervous system contain multiple myelin Ag-restricted and nonrestricted cell populations with the potential to mediate tissue injury. Previous studies indicate that it is possible to generate MHC class I-restricted myelin peptide-specific cytotoxic CD8 T cells, and that human adult OLs express MHC class I molecules in vitro. The purpose of this study was to demonstrate that myelin basic protein peptide-specific CD8 T cells could induce OL injury. We generated CD8 T cell lines from six healthy donors and five MS patients, and all cell lines were HLA-A2 positive. The obtained CD8 cell lines induced lysis of HLA-A2- but not HLA-A3-transfected HMy2.C1R cells in the presence of myelin basic protein peptide 110-118. In the absence of exogenous peptide, the CD8 T cell lines were cytotoxic to HLA-A2 but not to non-HLA-A2 OLs. Cytotoxicity was blocked with anti-MHC class I-blocking Ab. These results support the postulate that autoreactive CD8 cytotoxic T cells can contribute to the tissue injury in MS.     

B70 antigen is a second ligand for CTLA-4 and CD28

The membrane antigen B7/BB1 (refs 1, 2) is expressed on activated B cells, macrophages and dendritic cells, and binds to a counter-receptor, CD28, expressed on T lymphocytes and thymocytes. Interaction between CD28 and B7 results in potent costimulation of T-cell activation initiated through the CD3/T-cell receptor complex. Discrepancies between results with anti-CD28 and anti-B7 antibodies have suggested the existence of a second ligand for CD28 and CTLA-4 (refs 3, 6-8). We have generated a monoclonal antibody, IT2, that reacts with a 70K glycoprotein (B70). B70 complementary DNA was cloned from a B-lymphoblastoid cell line library and encodes a new protein of the immunoglobulin superfamily with limited homology to B7. B70 is expressed on resting monocytes and dendritic cells and on activated, but not resting, T, NK and B lymphocytes. IT2 substantially inhibited the binding of a CTLA4-immunoglobulin fusion protein to human B-lymphoblastoid cell lines and, together with anti-B7 antibody, completely blocked CTLA-4 binding. Further IT2 efficiently inhibited primary allogeneic mixed lymphocyte responses. These findings indicate that B70 is a second ligand for CD28 and CTLA-4 and may play an important role for costimulation of T cells in a primary immune response.