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CE, cloning efficiency (calculated from percentage of wells positive in cloning plates). Longevity is expressed as a percentage of established clones (i.e., those counted as positive in calculating the CE) that survive to 20, 30, or 40 population doublings (PD) (106, 1 09, 1012 cells, respectively). Origins: CD3, mature peripheral T cells from: (young)—apparently healthy donors under 30 yr.; (old)—perfectly healthy SENIEUR-compliant donors over 80 yr.; (cent)—centenarian donors >100 yr.; (CML)—middle-aged donor with chronic myelogenous leukemia in chronic phase treated with interferon-a; CD34, positively selected lineage-negative hematopoietic progenitor cells from peripheral (periph) or umbilical cord (cord) blood (Pawelec et al., 1998).

CE, cloning efficiency (calculated from percentage of wells positive in cloning plates). Longevity is expressed as a percentage of established clones (i.e., those counted as positive in calculating the CE) that survive to 20, 30, or 40 population doublings (PD) (106, 1 09, 1012 cells, respectively). Origins: CD3, mature peripheral T cells from: (young)—apparently healthy donors under 30 yr.; (old)—perfectly healthy SENIEUR-compliant donors over 80 yr.; (cent)—centenarian donors >100 yr.; (CML)—middle-aged donor with chronic myelogenous leukemia in chronic phase treated with interferon-a; CD34, positively selected lineage-negative hematopoietic progenitor cells from peripheral (periph) or umbilical cord (cord) blood (Pawelec et al., 1998).

(CE) of >50%, suggesting that these particular cloning conditions are favorable for outgrowth of a majority of T cells. However, cloning mixtures of T cells result in most of the TCC obtained being CD4+. To obtain CD8 (or TCR1 and especially NKT) cells, it is necessary to presort or otherwise remove CD4 cells. Cloning from a mixture of CD4 and CD8 cells with a CE of >50% implies that almost all CD4+ cells are initially clonable (defined as accumulation of ca. 1000 cells from one original cell). The CD4 T cell repertoire of the starting population is therefore present more or less in its entirety. However, after 20 PD, when the clone size has increased from 103 to 106, about half of the clones have been lost (mean of 47% for cloning of young donor CD3 cells; see Table 66.1). By 30 PD, about another half is lost, so that only one quarter of the originally clonable cells is still present. However, at 40 PD (which theoretically represents a very large clone size of 1012 cells), although more clones have been lost, 15% of the original starting clonal population does still remain. These results indicate constant attrition of the T cell population at the clonal level, but with retention of something like 5% of the original CD4 repertoire up to 40 PD and with retention of very rare clones for considerably longer. Although difficult to establish, similar clonal attrition probably occurs in vivo as well, at least in infectious mononucleosis, perhaps with quite similar distributions of clonal longevities (Maini et al., 1999).

Comparisons of T cell CE from young or old donors, or other sources, may be informative. Our hypothesis was that T cells generated in situ from CD34+ hematopoietic progenitors would have greater longevities due to their lack of a previous T cell proliferative history (Pawelec et al., 1998). Reciprocally, we anticipated that CE of cells from the elderly would be reduced as would their clonal longevity, and that this would be more prevalent in less healthy donors than in those rigorously selected for good health. Finally, we predicted that T cells from younger donors but in situations of chronic in vivo antigenic stress, such as in cancer, also would have lower CE and longevities. However, none of these expectations was fulfilled (see Table 66.1). CE was not lower in the elderly than in the young, and neither was the state of health of the donor critical. The CE was equally high when the cells derived from a patient with chronic myelogenous leukemia, a donor putatively subjected to a high level of antigenic stress. CE was also similar in the extra-thymic T cell differentiation cultures employed. It has to be concluded that CEs of the T cells from these markedly different sources are nonetheless similar. Is this true for average and maximum longevity also? It seems so: very similar proportions of TCC achieve 20, 30, and 40 PD regardless of their origin. In addition, the maximum longevity of the longest-lived clone is also similar and very close to the Hayflick Limit of 50-60 PD. Overall, it seems that CE and longevities of T cells in culture are essentially identical regardless of whether the cells to be cloned are derived from progenitors, young or old donors, healthy or sick.

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