of tumor lasting from 2 to more than 13 months. The rIL-2 was administered at 1 x 105 
U/kg intravenously every eight hours until dose limiting toxicity occurred. All adverse 
events were attributable to rIL-2. 
In the current study by Morecki, S. and Rosenberg, S.A. et al 10 , purified CD4( + ) and 
CD8(+) cells were isolated from fresh human PBMC samples or bulk melanoma TIL 
cultures by a direct panning selection using AIS CELLector™ CD8 and CD4 Culture 
Flasks. Adherent cells were allowed to grow in the original separation CELLector™ 
Flasks for a short time period and were then harvested and cultured in rIL-2 containing 
medium. Proliferation rate, phenotypic profiles, and cytotoxic activity of the isolated cell 
subsets were assayed after long term growth. Phenotypic analysis revealed that almost 
all PBMC- and TIL-derived CD4 and CD8 cell subsets maintained their characteristic 
surface markers without overgrowth of irrelevant cells. Ninety-three (93) to ninety-nine 
(99) percent of the cells recovered from the flasks consisted of the target population 
(either CD4 or CD8). The viability was always above 90 percent. The purified CD4(+) 
and CD8(+) cell subsets were further studied after long term growth to determine the 
usefulness of this separation and growth procedure for basic research and for clinical use. 
The isolated subsets maintained their ability to proliferate, kept their phenotypic profiles 
and remained functionally intact after long term culture (>40 days in several samples). 
In summary, the techniques described by Morecki, et al can be used to study the 
therapeutic effects of subpopulations of T lymphocytes. 
Letessier et al" conducted a study to evaluate antitumor functions of T lymphocyte 
subpopulations in blood (PBL) and tumor draining lymph nodes (LN) of patients with 
squamous cell carcinoma of the head and neck. AIS CELLecator™ CD8 Cell Culture 
Flasks were utilized to selectively capture CD8(+) cells for subsequent culturing. The 
investigators were able to capture a majority of CD8(+) cells from LN which represented 
less than 10 percent of the total lymph node cells. These cells successfully expanded in 
culture with relatively minor contamination from CD4( + ) lymphocytes. The mean 
enrichment in CD8( +) lymphocytes was considerable (80 percent +. 23) in these cultures. 
The selected and expanded CD8( + ) lymphocytes from LN showed a more selective 
pattern of cytotoxicity, with little, or no cytotoxicity against K562 or Daudi targets. 
This method for selection, expansion, and infusion of CD8(+) and CD4( + ) TIL is 
similar to that used in a current clinical trial evaluating the effect of blood derived and 
expanded autologous CD8(+) cells against the Human Immunodeficiency Virus or 
Acquired Immune Deficiency Syndrome (AIDS) patients or AIDS-Related Complex 
(ARC) patients. Following leukapheresis, CD8( + ) cells are captured using the Applied 
Immune Sciences, Inc. (AIS) CELLector™ CD8 Cell Culture Flasks. The CD8(+) cells 
are expanded in vitro and then infused into the subject of origin in five escalating doses. 
Continuous intravenous rIL-2 is administered during the final CD8(+) cell infusion and 
is continued for five consecutive days. This study is on-going, and so far the CD8( + ) 
autologous cell infusions (selected and expanded) have occurred without adverse effects. 
Recombinant DNA Research, Volume 15 
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