Multiparameter Kinetic Analysis Of Killer Cell Initiation Using Separated Lymphocyte Immune Rna

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Degree Name

Doctor of Philosophy (Ph.D.)


Initial studies were performed using whole spleen cell Immune RNA (I-RNA) from lymphocytes exposed to EL-4. By treating nonsensitized C57Bl/6J spleen derived lymphocytes with EL-4 tumor cell directed xenogeneic extracted RNA we were able to monitor early changes in cellular DNA content by flow cytometric (FCM) analysis and 3H-thymidine uptake. RNA content was monitored by 3H-uridine uptake. These kinetic parameters were correlated with cell mediated cytotoxicity (CMC) which appeared as early as 8 hours after activation as measured by release of chromium-51 from labeled EL-4 target cells. FCM analysis, 3H-thymidine and 3H-uridine uptake data showed peak S phase activity at 72 hr.Immune RNA derived from EL-4 sensitized glass adherent (GADH) and non-adherent (NGADH) Hartley Guinea Pig (HGP) cell fractions was used to monitor the transfer of CMC. Unsensitized C57Bl/6J mouse lymphocytes were treated with the resulting CADH and NGADH extracted I-RNA at 100 ug/ml for 1 hour at 37(DEGREES)C. GADH I-RNA treated lymphocytes showed the most elevated cytotoxic activity which was statistically significant at 24 hours, peaked at 72 hours and fell off by 96 hours. The NGADH I-RNA treated lymphocytes showed no significant cytotoxicity until 48 hours after initial exposure and dropped off by 72 hours. The NGADH kinetics resemble the cytotoxic kinetics of whole spleen cell I-RNA as reported previously (J.Hist.Cytochem., 27:491, 1979). Control RNA preparations did not show significant stimulation over normal C57Bl/6J RNA and RNASE control values.C57Bl/6J syngeneic tumor cell lines, a melanoma (B-6) and a mammary adenocarcinoma (M-3560), were used as targets for GADH and NGADH treated lymphocytes. The cellular fractions of the I-RNA treated lymphocytes showed selective cytotoxicity toward the EL-4 target. The cytotoxicity toward the M-3560 and B-16 targets fell within the control range, indicating a specificity of transfer of cytotoxic effector cell function by I-RNA. Supernatant fractions of both the GADH and NGADH cell cultures in the presence of normal C57Bl/6J lymphocytes also proved to be selectively cytotoxic toward the EL-4. This could indicate that some component may be released into the supernatant after I-RNA stimulation.


Health Sciences, Immunology

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