whose recipient developed an EBV lymphoproliferation after a 2 antigen 
mismatched transplant. These cells were generated in Dr Rooney's laboratory 
by coculturing donor peripheral blood lymphocytes with irradiated donor 
lymphoblastoid cell line. The line was expanded by addition of IL2 and weekly 
restimulation with irradiated autologous lymphoblastoid cells. Phenotyping 
showed the line was CD3 + CD8 + and cytotoxicity assays showed the line 
killed autologous LCLs but not allogeneic LCLs. Killing could be blocked by 
antibody to Class I MHC antigens. 
As the recipient had progressive EBV lymphoproliferation despite therapy 
with antibodies to CD21 and CD24 and Interleukin 2, we obtained IRB 
emergency approval to administer EBV specific cytotoxic T cells. At the time 
of infusion she had pulmonary and renal nodules, paratracheal nodes resulting 
in respiratory compromise and bowel infiltration resulting in gastro-intestinal 
bleeding. She received 3 x 10 7 cells/m 2 with no adverse effects; in particular 
she did not develop GVHD. However her paratracheal nodes continued to 
grow and 4 days later she required local irradiation and steroid therapy. At 
this stage her parents wished her to have no further therapy and she was 
discharged home where she died. Autopsy showed bowel hemorrhage and 
perforation was the cause of death but also showed necrosis of her pulmonary 
and renal nodules. While any beneficial effects on the pulmonary and renal 
nodules cannot confidently be ascribed to the CTL, she did not experience 
any adverse effects from the infusion. These results offer further 
encouragement to the proposed prophylaxis safety study. 
3.8 Risks of Administering EBV Specific CTL 
The main risk of this adoptive transfer approach would be that the donor 
derived anti-EBV T cells would cross react with host alloantigens and cause 
GVHD. Such reactivity would be unlikely due to the method of generation 
of the CTLs, and we will screen the CTL lines to ensure specificity for EBV 
infected targets. No GVHD has been observed in the Seattle patients 
receiving cytotoxic T lymphocytes (25) nor in the St Jude patient detailed 
above. Although we will screen CTL lines for reactivity against other host 
tissues such as bone marrow fibroblasts and PH A blasts, there is no 
completely reliable in vitro assay for excluding this possibility. We will take 
several additional precautions to minimize this risk. First we will not 
administer CTLs until Day +45 when the highest risk for GVHD has passed. 
Any patient with GVHD of greater than Grade 1 will be excluded from the 
study at this point. Second we will administer CTLs in escalating doses 
starting at 10 7 cells/m 2 . This is a much smaller number of T cells than 
administered at the time of marrow infusion even with a T cell depleted 
marrow. Finally, should any patient develop GVHD the site will be biopsied 
and examined histologically to confirm the clinical diagnosis. In addition 
DNA will be extracted and examined for the neomycin resistance gene (see 
below) to determine if administered CTL were present in the biopsy. 
Recombinant DNA Research, Volume 1 7 
