de Louis Leij

Clinical characterization of non-small-cell lung cancer tumors showing neuroendocrine differentiation features.

In most cases of small-cell lung carcinomas (SCLC) phenotypic features compatible with a neuroendocrine differentiation status can be identified by monoclonal (MOC) antibody-based immunohistological procedures. Similar features can be recognized only in a minority of non-SCLC tumors. During a period of 30 months, all diagnostic non-SCLC biopsies (141 cases) were prospectively analysed for the presence of markers indicative for neuroendocrine differentiation. In 31% of all cases, such a presence could be noticed. Neuroendocrine differentiation (50% to 100% positive-staining tumor cells) was recognized more frequently in adenocarcinoma when compared to large-cell and squamous-cell carcinoma (chi 2 = 9.31, 2 degrees of freedom, P less than 0.01). To investigate whether the clinical behavior of these neuroendocrine non-SCLC cases mimics SCLC, a multivariate analysis for prognostic factors was performed. Among other prognostic factors, biopsies containing more than 50% positive-staining tumor cells with the MOC antibody-1 (MOC-1) were recognized as negative prognostic factors.

Expression of class II major histocompatibility complex antigens on alveolar epithelium in interstitial lung disease: relevance to pathogenesis of idiopathic pulmonary fibrosis.

To determine whether expression of class II major histocompatibility complex antigens on alveolar epithelium is relevant to the pathogenesis of idiopathic pulmonary fibrosis (IPF) lung biopsy specimens were investigated from nine patients with IPF with or without connective tissue disease, four patients with sarcoidosis, eight patients with lung disease of presumably infectious origin, and five controls. The alveolar epithelium stained strongly with anti-Ia (HLA-DR) or Leu 10 (HLA-DS) monoclonal antibodies, in eight of nine biopsy specimens from patients with IPF, in three of four biopsy specimens from patients with sarcoidosis, in all six biopsy specimens from patients with presumably viral, mycobacterial, or pneumocystic lung disease, but not in control lung tissue, nor in two biopsy specimens from patients with bacterial pneumonia. Mononuclear cell infiltrates consisted of T4 positive (helper/inducer) lymphocytes, predominantly present in a nodular arrangement in the interstitium, and T8 positive (cytotoxic/suppressor) cells, distributed equally in the interstitium and subepithelially or intraepithelially. T8 cells outnumbered T4 cells in six of nine biopsy specimens from patients with IPF, but in none of the biopsy specimens from patients with sarcoidosis or interstitial lung disease of infectious origin. Although the expression of class II antigens on the alveolar epithelium which is infiltrated by T8 cells in IPF is consistent with local presentation of autoantigens and an ensuing local immune response, class II expression is also present in interstitial lung disease of sarcoidosis and microbial infections: its role in the pathogenesis of IPF must therefore remain speculative.

Detection and isolation of antigen-specific B cells by the fluorescence activated cell sorter (FACS)

A method is described for the isolation of antigen-specific B cells from immunized and subsequently boosted mice. Antigen-specific B cells were stained by incubation with fluorescein isothiocyanate (FITC)-labelled antigen and then detected and isolated in a fluorescence activated cell sorter (FACS). Ovalbumin (OVA) and Helix pomatia haemocyanin (HPH) were used as antigens in this procedure, yielding relative amounts of antigen-FITC-binding lymphocytes of 0.9 +/- 0.4% and 3.5 +/- 3.1%. The FITC-positive cells were visible as distinct cell populations in the FACS-generated histograms. All antigen-FITC-binding cells were B cells, as shown by double staining with phycoerythrin-conjugated anti-mouse Ig In addition, as tested in a spot-ELISA, the sorted, antigen-FITC-binding cell population contained almost the entire population of antigen-specific antibody-producing B cells. However, sorting had a negative influence on the antibody production capability of the sorted cells. Through washing of isolated spleen cells in the procedure before labelling with antigen-FITC proved to be essential for the specific detection of antigen-specific B cells, since staining without prior washing resulted in antigen-FITC binding to all B cells. This nonspecific staining phenomenon was caused by the presence of antibodies, specific for the immunizing/boosting antigen, which were also present in the spleen cell suspension. These antibodies formed immune complexes with antigen-FITC and bound to Fc receptors present on all B cells, interfering in this way with any specific binding of antigen-FITC to sIg on the B cells.

CRYOPRESERVATION OF NEWLY FORMED HYBRIDOMAS

A new freezing technique is described which permits time-consuming protocols as a first screening of newly formed hybridomas. In this procedure complete 96 well clustertrays with growing hybridomas are cryopreserved after programmed freezing. This procedure has been successfully applied to a number of fusion protocols for which the objective was to obtain monoclonal antibodies against tissue specific antigens. To this end hybridoma supernatants were screened by an immunoperoxidase technique on frozen sections. Freezing of the hybridoma containing clustertrays permitted extensive screening and partial characterization of the previously collected supernatants. After subsequent thawing of appropriate wells, the hybridoma clones proved to be viable and usually no loss of antibody production was observed.

HLA-DR-expressing CD8bright cells are only temporarily present in the circulation during subcutaneous recombinant interleukin-2 therapy in renal-cell carcinoma patients

The effect of subcutaneous recombinant interleukin-2 (rIL-2) therapy on the “activation status” of peripheral blood lymphocytes (PBL) of 17 renal cell carcinoma patients was investigated in a longitudinal study. The expression of the activation markers HLA-Dr and CD25 on cytotoxic T cells, helper T cells, and natural killer (NK) cells, was analysed using two-colour flow cytometry of whole-blood samples. In addition, the ability of isolated PBL to proliferate in vitro in response to various stimuli was investigated. The absolute amounts of NK cells and HLA-DR-expressing NK cells increased continuously during the whole course of therapy. The absolute amounts of T cells and HLA-Dr-expressing T cells, however, showed an early increase only during the first 1 or 2 weeks of therapy, after which the absolute amounts of HLA-Dr-expressing T cells decreased. In particular, the absolute amount of HLA-Dr-expressing CD8bright+ T cells was significantly lowered in the second half of therapy. PBL collected on day 7 of therapy (post-cycle-1 PBL) showed, as compared to those collected prior to therapy (pretherapy PBL), a decreased proliferative response in vitro after stimulation with phytohaemagglutinin, concanavalin A, soluble CD3 mAb (WT32) or rIL-2. This decreased in vitro response of post-cycle-1 PBL was also reflected in a decrease in the percentage of CD8bright+ T cells expressing HLA-Dr in cultures with rIL-2 or CD3 mAb, in contrast to cultures of pretherapy PBL, which showed an increase of this percentage. We conclude that T cells are the predominantly stimulated subpopulation during the first 2 weeks of subcutaneous rIL-2 therapy. The significant decrease in the absolute amounts of HLA-Dr-expressing T cells in the peripheral blood during the second half of therapy may partly be explained by a decreased responsiveness to rIL-2, but a selective redistribution of HLA-Dr-expressing cells may also be involved.

Early scd8 plasma-levels during subcutaneous ril-2 therapy in patients with renal-cell carcinoma correlate with response

Plasma sIl-2R and sCD8 levels of 12 patients with renal cell carcinoma were determined before and during subcutaneous rIl-2 therapy. Patients with a complete/partial remission showed a significantly stronger initial increase of sCD8 compared to patients with stable disease or tumour progression.

Refinement of an indirect immunotoxin assay of monoclonal antibodies recognising the human small cell lung cancer cluster 2 antigen.

Monoclonal antibodies (Mabs) from the Second International Workshop on Small Cell Lung Cancer (SCLC) Antigens that recognise the cluster 2 SCLC-associated antigen mediated potent and selective cytotoxic effects in an indirect assay of immunotoxin cytotoxicity. In this assay, the NCI-H69 cell line was treated with each Mab at 4 degrees C, washed to remove unbound Mab, and then incubated at 37 degrees C in the presence of a fixed concentration, 1 x 10(-8) M, of the screening agent, sheep anti-mouse IgG-ricin A chain. The use of a fixed high concentration of screening agent led to a 300-fold overestimate of the potency of a cluster 2-directed immunotoxin, MOC-31-ricin A chain. In contrast, when the concentration of the screening agent was identical to the Mab concentration, a precise match to immunotoxin potency was obtained. MOC-31-ricin A chain selectivity inhibited the incorporation of [3H]leucine by the NCI-H69, SW2 and GLC-8 SCLC cell lines by 50% at a concentration between 3 x 10(-11) M and 3 x 10(-10) M, and by the NCI-H125 lung adenocarcinoma cell line at 7 x 10(-11) M, but exerted no selective toxic effects upon human lung and non-lung tumour cell lines lacking surface expression of the cluster 2 antigen.

Poor induction of interleukin-2 receptor expression on CD8bright+ cells in whole blood cell cultures with CD3 mAb. Implications for immunotherapy with CD3 mAb.

To induce better stimulation of T cells during recombinant interleukin-2 (rIL-2) therapy of renal cell carcinoma patients, pretreatment with low-dose CD3 monoclonal antibody (mAb) has been proposed. However, in our clinic, such a treatment did not induce additional activation of T cells. To investigate this we performed whole blood cell cultures with rIL-2 or CD3 mAb as a stimulant. Cultures using isolated blood mononuclear cells were used as a control. When stimulated by the addition of rIL-2, the lymphocyte composition and activation of whole blood cultures did not differ from those of mononuclear cell (MNC) cultures. However, when stimulation was performed with CD3 mAb, CD8bright+ cells in whole blood cultures were not or only minimally induced to express CD25 or IL-2 receptor β (IL-2R). This is in contrast to the situation found in MNC cultures where all CD8bright+ cells expressed CD25 or IL-2Rß to a high extent at the end of culture. When rIL-2 or recombinant interferon γ (rIFNγ) was added to whole blood cultures together with CD3 mAb, significantly more CD8bright+ cells were induced to express CD25 or IL-2Rß. These results suggest that whole blood cultures represent the in vivo situation better than MNC cultures. In addition, the results suggest that, also in vivo, administration of low-dose CD3 mAb alone might not be sufficient to induce IL-2R expression on CD8bright+ cells, and would therefore not induce additional specific T cell activation in rIL-2-based immunotherapy. The presented results suggest that in vivosimultaneous administration of rIFNγ or rIL-2 with low-dose CD3 mAb might induce better stimulation of CD8+ T cells than CD3 mAb only.