Human Gene Therapy Subcommittee - 7/30/90 
to-person variation of the experiments? What was the 
patient's immune status? What was the patient's ADA 
level? Were the patient's cells capable of in vitro 
blastogenesis in response to stimulation with 
phytohemagglutinin or tetanus toxoid, either with or 
without exogenous interleukin-2 (IL-2)? Was the 
patient immunized to tetanus toxoid? 
3. Figure 2 is not interpretable. 
4. How many cells were given to each animal? How long 
after the animals were transplanted were the results in 
Figure 1 obtained? Were the untransfected cells 
treated in parallel with the transfected cells? If 
they were not treated in a parallel manner, were they 
transplanted immediately? What assays were done to 
show that equivalent viability and biological 
reactivity were present in the transfected and non- 
transfected cells? 
5. Were the allo-specif ic and tetanus toxoid-specific T 
cell clones obtained by primary limiting dilution 
analysis, or were they first expanded in bulk culture 
and then cloned by limiting dilution analysis? If 
there was an original bulk culture, what is the 
evidence that the alloreactive and tetanus-specific 
clones are derived from multiple precursors rather than 
a single antigen reactive cell? Has T cell receptor 
(TCR) rearrangement analysis been done on the clones to 
show that they are heterogenous, rather than 
homogenous, in terms of their TCR? Even if the cloning 
was done as a primary cloning, the TCR analysis needs 
to be done to demonstrate that all of the tetanus- 
specific and alloreactive T cells are not derived from 
a limited number of progenitors. 
Did the clones have the HLA antigens of the patient or 
mother? 
Were tetanus toxoid-specific clones directly clonable 
from the patient's peripheral blood? If tetanus 
toxoid-specific clones were produced from the patient's 
peripheral blood, the cloning of the antigen-specific 
clones from the SCID mice would be expected. The 
presence of the ADA gene in the clones does not prove 
that the ADA gene is required for the antigen-specific 
function. 
[62] 
Recombinant DNA Research, Volume 14 
