B. Mixed hemadsorption assays for IgG and IgM antibodies directed cell 
surface antigens (23-27). Specificity of responses to MHC antigens 
will be tested using autologous AV Epstein-Barr virus transformed B 
lymphocytes or AV fibroblasts. 
9.2 Cellular immune responses 
In selected patients where autologous tumor cells are available, MHC- 
restricted cellular immune responses can be evaluated. An attempt will be 
made to enter patients who have established cultures of autologous melanoma 
cell but these are not available for most patients. Although cellular immune 
responses are a critical measure of immune responses to melanoma, 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; c) evidence of cytotoxic or 
helper responses are not evident without in vitro stimulation by autologous 
tumor cells. This requirement makes it difficult to discern T cells that have 
responded to tumor from precursor T cells that are capable of recognizing 
tumor without responding in vivo. 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 units/ml and 
autologous or allogeneic stimulator cells as described for 7-14 days (reference 
51). The following assays to assess cellular immune function 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-MEL-29) 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 during co- 
cultivation with target cells. Interleukin-2 production is 
measured by enzyme-linked immunoassay (ELISA) and 
stimulation of the reference CTL cell line. Interferon -7 
production is measured by ELISA. Release of additional 
cytokines can be measured by ELISA, including interieukin-4, 
interleukin- 6 , tumor necrosis factor-a, and granulocyte- 
monocyte colony stimulating factor. 
B. Cytotoxic T cell assay. 
Cytotoxicity is measured in either a 4 hour or 18 hour 51 -chromium release 
assay (as described in reference 51). 
Recombinant DNA Research, Volume 15 
[901] 
