Malcolm G. Baines

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. IX. The quantitation of natural killer cell activity

On analysis ofin vitro assays of human natural killer (NK) cell function the inadequacy of commonly used methods of expressing lytic activity was apparent. A comparison was made of the data obtained using modifications of two equations—the simple exponential fit and the von Krogh equations. Both of these equations were found to satisfy the following essential criteria for use in these assays. First, the majority of the results obtained in the chromium-release assay could be used in data reduction; second, the resultant “dose-response” curve was reduced to linearity; and third, a single numerical expression was obtained which was directly proportional to the cytotoxic activity. Of the two methods the more conventional exponential fit was found to be the simpler to use. The closeness of fit of the experimentally derived data to the ideal curves did not support the possibility that normal lymphocyte preparations contain suppressor cells capable of inhibiting NK activity. Data have also been presented showing that NK-sensitive targets could be categorized with respect to their susceptibility by comparing the slopes of the target cell survival curves obtained using the exponential fit equation. These observations are relevant to the accurate assessment of NK activity in patient populations and to the determination of the effects of disease and its treatment on this activity.

Immunofluorescence using dichlorotriazinylaminofluorescein (DTAF) I. Preparation and fractionation of labelled IgG

Dichlorotriazinylaminofluorescein (DTAF), the product of the reaction of aminofluorescein with cyanuric chloride, is an effective reagent for conjugating fluorescein to immunnoglobulins. DTAF has absorption and emission properties nearly identical to fluoresceinisothiocyanate (FITC) and DTAF and FITC-labelled antibodies are similar in terms of preparation and specificity of immlnofluorescence. However, DTAF is superior to FITC with regard to cost, purity and stability. Also, DTAF-labelled rabbit IgG conjugates can by quickly and efficientyly fractionated by simple ammonium sulfate precipitation procedures to yield preparations free of both over- and under-conjugated material. In addition, over-conjugated protein can be readily removed from DTAF:IgG conjugates by appropriate adjustment of pH and temperature.

Quantitation of a whole blood assay for human natural killer cell activity

A method is described for the measurement of human natural killer cell activity using heparinized whole blood in a 51chromium release assay. Fractionation-reconstitution experiments showed that the cytotoxic activity was abolished by removal of the Fc receptor bearing lymphocytes, but not by the elimination of monocytes and granulocytes. Autologous or pooled plasma was not found to possess inherent cytolytic activity. By analogy to an enzyme kinetic reaction, the results were expressed as kinetic lytic units (KLU) which were defined as the maximum number of target cells that could be lysed per unit time per milliliter of whole blood. The buffy coat cytotoxicity (BCC) assay is quick, easy to perform, and suitable for screening and monitoring of natural cytotoxicity. Since this methodology preserves the actual concentration of natural killer cells, it may represent a truer reflection of in vivo events.

Studies of human natural killer cells. III. Neutropenia associated with unusual characteristics of antibody-dependent and natural killer cell-mediated cytotoxicity

A 52-year-old Caucasian man with chronic neutropenia and recurrent infections was found to have an increased proportion of peripheral T lymphocytes having Fc receptors for IgG (T(γ). Although levels of antibody-dependent cell-mediated cytotoxicity (ADCC) and “natural” killing (NK) by unfractionated lymphocytes were similar to those of a control donor, the frequency of NK cells was markedly increased. Removal of E rosette-forming cells eliminated both NK and ADCC by the patients peripheral blood, in marked contrast to theenhanced cytotoxicity seen with control lymphocytes. Both normal and patient ADCC and NK functions were removed by depletion of Fc receptor-bearing cells. These depletion experiments proved that all of the patients killer cells were E rosetteforming Tγ cells, in contrast to the heterogeneous pattern of Nullγ and Tγ killer cells seen in the blood of normal donors. The homogeneity of the Tγ proliferation suggested that ADCC and NK were mediated by the same cell type, albeit acting by different mechanisms. The addition of the patients serum and lymphocytes to chromiumlabeled normal granulocytes caused a low but significant level of cytotoxicity, indicating that the patients neutropenia may have been caused by a similar mechanismin vivo. There was no evidence of complement-dependent serum antibody-mediated neutrophil lysis, but one serum sample taken over the course of the patients disease agglutinated granulocytes from four of five donors tested.

Conjugation of DNP-, TNP- and dansyl haptens to carbohydrates and erythrocyte membranes via dichlorotriazine derivatives☆

Abstract Stable, soluble labelling compounds are formed by the reaction of DNP-, TNP- and dansyl-L-lysine with cyanuric chloride at 0°C in aqueous solutions. These compounds react efficiently at room temperature with carbohydrates and with sheep erythrocyte membranes. Mice immunized with Ficoll haptenated with these reagents yield characteristic thymus-independent plaque-forming cell responses.