MLR, mixed lymphocyte-tumor Rx., weird result

John Ladasky jladasky at pmgm.Stanford.EDU
Tue May 26 19:06:25 EST 1998

Greetings, fellow immunologists (and pretenders like Yours Truly),

	I am combing through some literature regarding mixed lymphocyte-
tumor reactions (MLTR) and allogeneic mixed lymphocyte reactions (MLR).
There's a lot of it.  I am hoping that a knowledgeable person out there
will be able to help me narrow down my reading list.  It would be really
nice to find a review which compares and contrasts the two assays, or
something similar.

	I am attempting to identify any effects that mutations in beta-
2 microglobulin might have on CTL recognition.  I have stably transfected
a beta-2m negative cell line (Daudi) with wild-type beta-2m, and with
three mutants.  I then used these cells as targets in mixed lymphocyte-
tumor reactions with an allogeneic donor (me).  After two weeks of stimu-
lation, the plan was to isolate CD3+CD8+ cells and establish CTL lines by
limiting dilution.  Before I even got to this, I was surprised to see
gross differences in the primary cultures.  After only three days, some
of the cultures had many blastocytes, and others did not.  Even after 14
days, the cultures that had proliferated moderately had not done much.  I
am also seeing surprising differences in the frequency of lines that I am
growing out.  Here's a summary:

     Irradiated		  Primary	      Number
  Stimulator cells	culture growth	  of lines growing
 untransfected Daudi	  minimal		ND
 Daudi/wild type b2m	   strong		 6
 Daudi/b2m mutant 1	   strong		 0
 Daudi/b2m mutant 2	  minimal		11
 Daudi/b2m mutant 3	   strong		 1
 allogeneic PBMC	  minimal		ND

	As I said, first I was surprised that strong growth was observed
in the first few days in some cultures.  I don't see this with allogeneic 
PBMC under the same conditions.  Second, the two mutants of beta-2m that
did induce strong primary proliferation are giving me *almost no* CTL
lines -- whereas the wild-type beta-2m gave me both proliferation and
lines -- and, stranger still, the mutant which apparently inhibited 
primary proliferation is giving me the most lines!  Three mutants, three

	I did a flow cytometry analysis of the bulk cultures at day 14,
at the same time that I sorted the CD3+CD8+ cells and established the
limiting dilution cultures.  Every cell type seems to have proliferated
about evenly in the cultures that grew.  If anything, the NK cells grew
slightly more than the rest.

	These results make me wonder about non-specific proliferation in
MLTR.  It seems that the cultures that grew a lot were expanding *all* 
lymphocytes, whether or not they were antigen-specific.  This might 
explain why mutants 1 and 3, which gave me a lot of primary prolifera-
tion, gave me fewer lines.  A colleague in our lab sees this same non-
specific proliferation of cells when we use transfectants of the class
I-negative, EBV derived line 721.221.  This even occurs with syngeneic
effectors.  Since it has been an apparently uniform phenomenon in her
class I transfectants, she views this as nothing more than an obstacle
in the way of cloning CTL.  I probably would, too, if I hadn't seen a
differential phenotype.  Is it something special about tumor cells?  Or
about B cell-derived lines?

	Obviously, I need to repeat these results, and this time I will
include control CTL clonings with untransfected Daudi and allogeneic PBMC
targets.  I will also try a second donor of effector cells.  I'm also
thinking that I should try something like using purified allogeneic B
cells in one MLR experiment -- there are a lot of different cell types
in the primary allogeneic PBMC.  The Daudi stimulators are uniform, and
Daudi is a Burkitt's lymphoma (B-cell like).

	I may also want to try some additional experiments such as 
measuring 3H-thymidine incorporation and cytokine analysis.  However, I 
definitely need to know *what's worth looking for* before I design a 
whole bunch of useless experiments.  If you can point me in the right
direction, you will of course be acknowledged in the paper for "helpful

Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw.					- John Ladasky

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