July 30, 1990, Human Gene Therapy Subconmittee 
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. 
6. What degree of infection was obtained when cells were treated with a 
single cycle of virus, which is equivalent to the proposed NIH protocol? 
7. Figure 6. Why is there no diagnostic band in clone B56 at 5.3 kilodaltons? 
8. Figure 2. Where are the controls? Where is the histogram showing 
staining with directly labeled ascites to demonstrate that the 4% staining is 
CD4-specific? What is the staining pattern with an antibody to CDS? 
How many CD4^ cells were detected when non-transfected cells were 
injected? How many CD4'^ cells were detected when the peripheral blood 
T lymphocytes, transfected with a control vector, were injected? 
9. Was human antibody to tetanus toxoid produced in addition to just 
immunoglobulin, since tetanus toxoid-specific T lymphocytes are present? 
Dr. Parkman summarized these questions as falling into three general areas: 
1. What is the origin of the immunologially functional cells? 
2. Was the fact that immunologically functional cells were present due to the 
fact that the ADA gene was inserted, or could an analogous degree of 
function be seen in the patient's peripheral blood? 
3. If the immunological function seen was a consequence of the insertion of 
the ADA gene, how diverse was the repertoire or spectrum of cells that 
were responding? Were they all derived from a limited number of cells or 
were they many different cells? 
Dr. Childress said he felt the conditions previously set for approval of the protocol were 
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