to the next infusion and were augmented to levels equivalent to healthy CMV 
seropositive donors by the third infusion (cell dose 3.3 x lOVm 2 ) One patient has now 
been followed for six weeks following completion of all four T cell infusions and still 
had detectable CD8 + CMV-specific T c responses but had not yet developed a CD4 + 
CMV-specific T H response. In prior studies, we have not observed the reconstitution 
of CD8 + T c in the absence of CD4 + T H [48, and unpublished data], suggesting the 
CD8 + T c responses persisting in this patient are due to transferred T cells. The 
remaining two patients had detectable but weak CD8 + CMV-specific T c responses and 
these were augmented during T cell therapy. Thus, these results demonstrate that 
CMV-specific CD8 + T c clones can be adoptively transferred safely to BMT recipients 
and persist in vivo as functional effector cells. 
D. Generation and Retroviral Modification of CD8 4 HIV- Specific T, 
Based on the encouraging results of adoptive immunotherapy for reconstituting CMV-specific 
immunity, it should be possible to reconstitute or augment HIV-specific immunity for HIV 
seropositive individuals undergoing allogeneic bone marrow transplant. Culture methods to 
generate CD8 + HIV gag-specific T c from HIV seropositive individuals have been described 
[20-26] and are operative in our laboratory. T cell clones with specificity for the HIV gag 
and envelope gene products have been generated from four consecutive HIV seropositive 
individuals (unpublished data). These clones can be expanded in long term culture to 
numbers sufficient for adoptive immunotherapy studies. Due to the increased likelihood of 
mutation in the HIV envelope potentially resulting in loss of T cell epitopes, we propose to 
use T c with specificity for the gag gene product for adoptive immunotherapy. In contrast to 
the CMV immunotherapy study, there are several unique considerations in the proposed HIV 
immunotherapy study. Firstly, due to the nature of host cells potentially infected with HIV, it 
is possible the recognition and elimination of infected cells by transferred CD8 + T c might 
induce unique toxicities in these patients. In particular, HIV can infect monocyte-derived 
microglial cells in the central nervous system (CNS) [52] and alveolar macrophages in the 
lungs [53], and an inflammatory response at these sites induced by cytokine release from 
transferred CD8 + T c could result in CNS toxicity or lymphocytic alveolitis, respectively. 
Although controversial, it has been suggested that marrow progenitors are susceptible to HIV 
infection [54,55], and should donor hematopoietic progenitors become infected by HIV, their 
elimination by CD8 + effector cells could compromise engraftment. Thus, for HIV, as well as 
potentially for treatment of other diseases, it would be desirable to have a means of selectively 
eliminating transferred T c clones if toxicity occurs. Secondly, the T cells to be used in this 
study will be derived from the host and are potentially susceptible to immunologic rejection 
by engrafted donor lymphocytes due to recognition of minor histocompatibility antigens. 
Thus, it would be desirable to have a sensitive means of determining the duration of in vivo 
persistence of transferred T c since this would be expected to impact on potential antiviral 
efficacy, and this data could be used to guide the development of protocols utilizing 
alternative infusion regimens. 
A solution to these problems may be provided by using retrovirus mediated gene transfer. 
We have investigated using a retroviral vector encoding a novel selectable marker 
incorporating the hygromycin phosphotransferase gene and the herpes virus thymidine kinase 
(HSV-TK) gene to provide both a marker gene and a suicide gene in T cell clones. The 
HSV-TK, in distinction to mammalian TK enzymes, can efficiently phosphorylate nucleoside 
analogues such as ganciclovir, thereby rendering transduced mammalian cells susceptible to 
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