Anne S. Hamblin

A method of preparing blood leucocytes for flow cytometry which prevents upregulation of leucocyte integrins

LeuCAM (CD11/CD18) cell-surface antigens are easily upregulated on cell manipulation ex vivo. A procedure for preparing leucocytes, in which human blood is immediately treated ex vivo with buffered formaldehyde and then the erythrocytes and platelets are removed by lysis and differential centrifugation, has been successfully applied to the analysis of LeuCAM antigen expression by flow cytometry. We show that the increased expression of monocyte CD11/CD18, which occurs when mononuclear leucocytes are separated by a standard Lymphoprep density gradient separation, can be avoided if cells are fixed immediately. Following this fixation polymorphs are unable to upregulate CD11/CD18 in response to fMLP stimulation in vitro. The technique produces lymphocyte, polymorph and monocyte populations that can be clearly defined on the basis of forward scatter and side scatter, and preserves the expression of various surface antigens; the percentages of gated lymphocytes expressing CD3, CD4, and CD8 were similar to those obtained using a commercial fixing and lysis solution. The processing does not render cells permeable to antibodies, as evidenced by our failure to stain cells with antibodies to intracellular antigens. We believed the method to be useful for measuring CD11/CD18 expression on blood leucocytes from normal or pathological specimens and to have application to the measurement of other cells surface antigens which may also be upregulated by the separation procedures.

Endothelial cell presentation of antigen to human T cells.

Activation of human T cells requires presentation of antigen by Ia (HLA-DR in man) bearing cells of the mononuclear phagocytic series (macrophages, MO, and more recently Langerhans cells, dendritic cells, and vascular endothelial cells. Since T cells must cross endothelial barriers to enter extravascular tissues during immune reactions, we investigated the role of endothelial cells in antigen presentation. Endothelial cells were cultured from human umbilical veins and identified by classic morphology and specific markers (factor VIII related antigen, and so on). Antigen-pulsed endothelial cells were used to present antigen to MO-depleted human T cells; activation was assessed by 3H-thymidine uptake. The HLA-DR compatible endothelial cells were as effective as MO in reconstituting MO-depleted T-cell responses. The endothelial cell reconstituted responses were antigen specific, HLA-DR restricted, and blocked by monoclonal antibodies to HLA-DR framework structures. Moreover, the T-cell responses were clonal with respect to HLA-DR. A monoclonal antibody completely eliminated MO reconstitution of the MO-depleted response without diminution of endothelial cell reconstitution of the same response. Fibroblasts and smooth muscle cells cultured from the same umbilical veins could not reconstitute the MO-depleted T-cell response. These data indicate that endothelial cells play an important and distinctive role in lymphocyte triggering.

LONG-TERM SURVIVAL AND PRESERVATION OF NATURAL-KILLER-CELL ACTIVITY IN A XERODERMA-PIGMENTOSUM PATIENT WITH SPONTANEOUS REGRESSION AND MULTIPLE DEPOSITS OF MALIGNANT-MELANOMA

Summary A 67‐year‐old man with xeroderma pigmentosum (XP) originally presented with malignant melanoma at the age of 28 years. This recurred 22 years later and subsequently numerous primary and secondary melanomas developed on the skin, several of which underwent spontaneous regression. Despite a marked lymphopenia, the proportion of natural killer cells was elevated and it is proposed that this led to the regression of the melanomas. Skin‐derived fibroblasts from the patient were more sensitive to UVC (D10ε3 J/m–2) than those from normal individuals (D10– 15 J/m–2).The fibroblast culture was shown to be defective in excision repair with <10% of residual activity compared with controls. No assignment to a complementation group has yet been made. There was an elevated frequency of mutants resistant to 6‐thioguanine in the circulating T lymphocytes.

Antigen‐presenting capacity in normal human dermis is mainly, subserved by CDla+ cells

A proposed role for antigen‐presenting dermal dendrocytes in the pathogenesis of many dermal inflammatory skin diseases remains speculative. We therefore sought to determine the phenotype and functional characteristics of antigen‐presenting cells isolated from normal human dermis. Normal adult human skin was incubated overnight with dispase at 4°C, the epidermis was removed, and the residual dermal preparation was then minced and digested with a mixture of hyaluronidase, collagenase, and DNAase at 37°C, prior to filtration through mesh. Dermal cell suspensions thus obtained were stained using specific monoclonal antibodies, and analysed by fluorescence micro‐ scopy or flow cytometry. Mean values were as follows: CD45+ leucocytes 39%, HLA‐DR+ cells 39%, Ulex europaeus agglutinin I+ endothelial cells 26%, CD1a+ cells 3.9%, CD11b+ cells 16%, CDllc+ cells 6%. Mitomycin C‐treated crude dermal cell suspensions induced allostimulation of peripheral blood mononuclear cells in a 7‐day culture, as assessed by 3H‐TdR incorporation. Depletion of CDla+ Langerhans‐like cells from the dermal cell preparation, by 95, 74 and 90% in three separate experiments using immunomagnetic beads, reduced 3H‐TdR incorporation at optimal responder‐to‐ stimulator cell ratios by 90, 64, and 87%, respectively. Our findings suggest that, in normal human dermis, the great majority of the alloantigen‐presenting capacity resides in the CDla+ Langerhans cell‐like dendritic antigen‐presenting cell population, and not to any great extent in either CDla− macrophage‐like cells, or HLA‐DR+ endothelial cells. The relationship of the CDla+ dermal antigen presenting cells to the Langerhans cell lineage remains to be determined.

The Role of Cytokines in Asthma

Cytokines, which are proteins released by cells, play essential roles in specific immune responses and inflammatory processes.Id There is ample evidence that inflammation of the airways leads to bronchial hyperresponsiveness which is characteristic of asthma.j In addition, extrinsic asthma in atopic subjects is accompanied by immediate (Type I) hypersensitivity to allergens and this occurs when there is an IgE immune response specific for the sensitising agent.5 The occurrence of both inflammation and specific immunity in asthma makes it likely that cytokines pIay a significant role in the development of this disease.

Molecular cloning lends credence to lymphokine research

There is no longer any doubt that lymphokines are real. At a recent meeting some major advances in lymphokine research were clear - most notably, the application of molecular cloning techniques which have given credence to lymphokines and provided the necessary homogeneous materials for in-vivo and in-vitro studies. Clarification of the structure and function of some lymphokines has diminished the use of assay-related acronyms to describe ill-defined activities and evidence is accumulating for cascades of lymphokines and synergy between molecules. The emerging picture is exciting but complicated.

Cell-mediated immunity: Correlation of mixed-leucocyte-macrophage migration inhibition with delayed-type hypersensitivity after immunization and donor-specific transfer of cell migration inhibition by dialyzable leucocyte extract☆

Active and adoptive sensitization of rhesus monkeys (Macacca mulatta) as well as the development of a novel sensitive in vitro cell migration inhibition assay for cell-mediated immunity (CMI) in this species are described. First, the correlation of mixed leucocyte-macrophage migration tests (LMMI) with the whole blood lymphocyte transformation (LT) and the delayed hypersensitivity skin test (DH) in immunized animals are shown. Second, these tests are used to demonstrate adoptive transfer of specific/nonspecific cellular immunity (CMI) with dialyzable leucocyte extract (DLE) from immunized donor to unimmunized recipient monkeys. Seventeen animals were immunized with keyhole limpet haemocyanin (KLH) or hepatitis B surface antigen (HBsAg) in Freunds complete adjuvant (FCA) or with FCA alone. Acquisition of antigen-specific cell-mediated immunity was detected by all three tests within 5 weeks of immunization. Positive LMMI responses were associated with positive DH and LT. However, there was no correlation between the magnitude or time of development of the three responses. Therefore, the LMMI test, like the LT test, is an in vitro parameter of DH, but reflects the activity of different subpopulations of lymphocytes and is regulated by different mechanisms. In addition, 12 naive animals received DLE. Within 3 weeks, transfer of sensitivity was detected towards antigens to which the recipients had previously not been reactive but the donors had been. An enhancement of transformation response to phytohaemagglutinin was also seen. Thus, rhesus DLE contains both donor-specific transfer factor-like and nonspecific adjuvant-like activities. In DLE recipients, unlike immunized animals, LMMI responses were dissociated from DH or LT responses in that positive LMMI was mostly seen with negative DH or LT to antigens. Therefore, LMMI emerged as the most sensitive assay for detecting adoptive transfer of CMI by DLE in vivo, supporting the view that different mechanisms regulate LMMI, LT, and DH.

Normal cellular immunity in Cockayne's syndrome: evidence for the role of defective immunosurveillance in the causation of skin cancer

One hundred and twenty-one patients who had received a renal allograft between 4 months and 21 years previously (mean±SD 71162 months) were studied. Seventy-two patients were conventionally immunosuppressed with azathioprine and prednisolone, 36 had been given the current regimen of cyclosporin, azathioprine and prednisolone and 13 had received other combinations of drugs. Forty-five patients had viral warts, of whom 20 had more than 10 warts. The presence of viral warts was significantly associated with pale skin type, excess sun exposure and with duration of allograft. Viral warts were significantly more common in those on conventional immunosuppressive therapy, but this could be solely a reflection of the difference in duration of transplant between the two groups. Seven per cent had fungal skin infections which were significantly more common in patients with skins that tanned easily than in those who burned in the sun. Twelve patients were found to have developed dysplastic or neoplastic skin lesions since transplantation. The incidence of dysplasia increased with increasing age and was significantly associated with pale skin type, excess sun exposure and duration of allograft. Despite the shorter duration of treatment in th ose on the new treatment regimen, there was no difference between the two groups in the proportion of patients with dysplastic skin lesions. Immunosuppression-related skin disease may be a significant problem in allograft recipients in this covmtry and we suspect that patients taking cyclosporin will have similar problems to those on conventional immunosuppressive drugs alone.

Lymphokines and the Lymphoendothelial System : An Illustration of Immunoregulatory Integration

The possibility that non-antibody products of lymphocyte activation might be involved in the expression and regulation of lymphocyte function arose indirectly from consideration of the role of lymphocytes and macrophages in experimental delayed hypersensitivity (Dumonde, 1967; Dumonde et al., 1968). Analysis of experimental cell-mediated immune phenomena had pointed to the role of cellular interactions between a few specifically sensitized lymphocytes and the majority of other host cells participating in these responses (see Turk, 1967). These features were consistent with the concept that special ‘mediators’ of cellular immunity could well facilitate such interactions; and that this would bring the cellular immune system into line with other biological systems in which differentiated cell products (for example endocrine and neurotransmitter substances) were known to mediate complex physiological events.

The Response of Tumor-Bearing Patients to the Injection of Lymphoid Cell Line Lymphokine

We began this work in 1976 at a time when lymphokines were becoming accepted as mediators of cellular immune responses and when consideration was being given to the immunodepression associated with progressive cancer and with its treatment. At that time, work on leukocyte interferon, dialyzable leukocyte transfer factor, and thymic extracts had established a precedent for the therapeutic investigation of biological materials in patients with otherwise irreversible neoplastic disease; and it seemed that lymphokines could also have therapeutic potential in human cancer (Hamblin et al., 1978). We were examining lymphokines generated by the cultured B-lymphoblastoid cell line RPMI 1788 as potential standards in the leukocyte migration test (see Hamblin et al., 1982); and when Papermaster et al. (1976) reported on the safe intralesional (i.l.) injection of RPMI 1788 lymphokine into human cutaneous metastases, we decided to use this source of lymphokine for the present study. With ethical permission and informed consent, we set out to extend knowledge of the histopathological responses to the i.d. and i.l. injection of RPMI 1788 lymphokine (“LCL-LK”) in patients with advanced cancer and to investigate the clinical, hematological, biochemical, and immunological responses to single and repeated i.v. injections of LCL-LK.