«In vitro» Cytotoxic Activity of Normal and Leukemic Anti-Leukocyte Murine Sera

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Abstract
These experiments were designed to evaluate the specificity of cytotoxic antibodies for normal and leukemic leukocytes from mice with different genotypes. C57BL and C3Hf/Gs mice were immunized with allogeneic splenic cells from C3Hf/Gs, C57BL and (C3Hf/Gs x CBA T6T6) F1 donors. The animals were injected intraperitoneally once a week for 6–7 weeks and serum was obtained 7 days after the last injection. The cytotoxic effect of the sera on leukocytes was evaluated using the Terasaki test, which is based on direct phase contrast observation of refraction modification in damaged cellular elements. By using donors and recipients with dissimilar H-2 locus, an increase in the cytotoxic index was observed which was related to the number of immunizing injections. Values were fairly uniform for the different combinations tested. The maximum antibody titer was obtained 7 days after the last injection with notable decrease after 14 and 30 days. Cross tests between antisera and leukocytes with the same or different H-2 allele have demonstrated a high specificity of the isoantisera for the different H-2 locus alleles (k, b). In another group of experiments, the effect of isoantisera on leukocytes from animals with generalized leukemia was tested. The leukemia was induced by inoculation at birth with either Graffi or Gross virus. In the 12 cases studied, the cytotoxic effect on leukemic cells was always less than on normal cells of the same strain. Finally, a group of experiments was carried out using leukemic anti-leukocyte sera obtained by immunizing C57BL animals with cells derived from 2 Graffi virus-induced leukemias grafted into (C3Hf/Gs x CBA T6T6) F1 mice. These sera presented maximum cytotoxic index values on leukocytes of (C3Hf/Gs x CBA T6T6) F1 animals with Graffi virus-induced leukemia. Lower values were observed for normal leukocytes of the same strain. (C3Hf/Gs x CBA T6T6) F1 cells originating from Gross virus-induced leukemia were less sensitive to these sera, not only in comparison with Graffi virus-induced leukemia leukocytes but with normal (C3Hf/Gs x CBA T6T6) F1 leukocytes as well. It was also possible to demonstrate positivity in the cytotoxic response with C57BL leukemic leukocytes (Graffi virus-induced). These data seem to indicate that a reduction in the histocompatibility antigens of the leukemic leukocyte occurs contemporaneously with the appearance of tumor-specific antigens, and both phenomena are most likely related to neoplastic transformation. The following hypotheses are discussed as possible explanations of these phenomena: the possibility of true antigen deletion; the spreading out of membrane antigenic receptors as a consequence of increased cellular volume; the appearance of the TL antigen in the leukemic cells causing a partial loss of H-2 antigens.