9.0 EVALUATION OF IMMUNE RESPONSE 
9.1 Serologic studies 
The most direct method to measure immune response to renal carcinoma is 
detection of antibodies. The detection of IgG antibodies induced by immunization is 
also a possible indirect measure of helper T cell responses, because induction of 
high affinity, mature IgG responses requires T cell help by CD-4 positive T cells. 
All patients will have peripheral blood sera obtained for serologic analysis of 
antibodies directed against the SK-RC-28 and SK-RC-39 cell lines (to include 
gpl60, gpl20, gpl40, and Uro-8) and autologous tumor cells whenever available. 
In addition, a control for positive immune response with antibodies against 
allogeneic MHC class I and II antigens expressed by SK-RC-28 and SK-RC-39 will 
be done. It is expected that most patients will develop strong IgG responses against 
class I and class II alloantigens expressed by the two cell lines. Antibodies against 
cell surface and intracellular antigen will be detected by: 
A. Immunoprecipitation of 1% NP40 lysates of SK-RC-28 and SK-RC- 
39 metabolically labeled with 35 'methionine. 
B. Mixed hemadsorption assays for IgG and IgM antibodies directed 
against cell surface antigens (as stated above). 
9.2 Cellular immune response 
In selected patients where autologous tumor cells are available, MHC-restricted 
cellular immune responses can be evaluated. Although cellular immune responses 
are a critical measure of immune response, there are the following limitations: a) 
autologous tumor cells are necessary to detect responses, b) autologous normal cells 
and MHC-matched allogeneic cells are necessary to evaluate the specificity of 
responses, and c) evidence of cytotoxic or helper responses are not evident without 
in vitro stimulation by autologous tumor cells. Mononuclear cells from peripheral 
blood will be purified by Ficoll-Hypaque gradient centrifugation. Cells will be 
cultured with autologous tumor cells, interleukin-2 (20-100 u/ml) and autologous or 
allogeneic stimulator cells as described for 7-14 days (reference 51). The following 
assays to assess cellular immune response will be done when autologous tumor cells 
are available: 
A. Helper T cell Assays. 
Peripheral blood lymphocytes will be cultured with autologous 
tumor cells, autologous normal cells and allogeneic tumor cells 
(e.g. SK-RC-28 and SK-RC-39) for 48 hours. Proliferation will 
be measured by uptake of 3 H-thymidine in a 4 hour pulse at the 
end of the assay. Additional measures are secretion of cytokines 
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Recombinant DNA Research, Volume 15 
