Graham Pawelec ¹, Ed Remarque ², Yvonne Barnett ³, Rafael Solana ⁴

1 University of Tübingen, Tübingen, FRG ², University of Leiden, Leiden, Holland ³, University of Ulster, Coleraine, Northern Ireland ⁴, University of Córdoba, Córdoba, Spain

Received 12/29/97 Accepted 1/5/97

6. CULTURE MODELS FOR IMMUNOSENESCENCE: THE HAYFLICK LIMIT APPLIES TO NORMAL T CELLS

Since the T cell response requires waves of clonal expansion followed by contraction and re-expansion on recontact with antigen, limits to the proliferative capacity of the T cell clones might impact deleteriously on the overall response. T cell clones in vitro may provide a good model for investigating age-associated changes in a longitudinal manner. They can be maintained for extended periods in tissue culture, but in most cases they have finite lifespans, eg. see refs. (94,154,166-172). In our comparison of the in vitro longevity of T cell clones (TCC) derived from mature peripheral T cells and those derived from T cell progenitors of the same donor we found that both manifested limited lifespans. Progenitor-derived cells survived on average 20 populations doublings (PD) longer, but this corresponded to the length of time that they required to differentiate from progenitors into cells with a mature T cell phenotype (173).

Could there be exceptions to T cells with finite lifespans? Certain human T cell clones which have been cultured for many years must be presumed to have exceeded the Hayflick limit, although this has not been formally measured by any of the investigators working with such clones. How can these potential discrepancies be resolved? Human T cells infected with HTLV-1 or Herpes saimiri virus can become immortalized. In the latter case, the cells retain a normal functional phenotype, ie. they remain dependent upon exogenous growth factor for their continued proliferation, and they still respond specifically to stimulation via their antigen receptor and non-specifically via the alternative activation pathway (CD2/CD58-dependent). Therefore, inadvertant infection with H. saimiri would result in retention of apparently normal immunological attributes coupled with indefinite lifespan. However, the chances of inadvertant infection with this non-human virus are presumably very low, and can be excluded by screening for known viruses. Nonetheless, there always remains the possibility that rare events featuring transformation with unknown viruses might account for some examples of apparent immortality of TCC.

It may still be questioned whether suboptimal culture conditions for T cells are responsible for the short lifespans of the majority of TCC. Since the rare long-lived TCC are cultured under apparently very similar conditions to the normal, short-lived, ones, this may seem a priori unlikely. However, some simple manipulations of tissue culture conditions may be sufficient to affect longevity, and for some reason certain rare T cells might more successfully adapt than others. For example, it has become apparent that simply reducing the oxygen content of the culture environment from the supraphysiological tension commonly employed (air) to a more physiological level can result in considerable lifespan extension of fibroblasts (174,175). Whether this is the case for T cells and whether they are more sensitive to oxidative damage, has not yet been reported. Other possible manipulations which have been reported to extend the lifespan of fibroblasts, but which have not been tested on T cells, include using hydrocortisone, carnosine, anti-sense oligonucleotides for p53 and Rb, high albumin concentrations, additional growth factors and other hormones (reviewed in ref. (176)). Optimisation of culture conditions using capillary bed culture cartridges may also better mimic the in vivo environment and lead to extended lifespans, but this has also not yet been demonstrated (177).

Most human TCC are generated from cells obtained from peripheral blood, but such recirculating cells may not be truly representative of the T cell pool. The major lymphoid organs from which T cells can be obtained are skin and gut. T cells infiltrating the skin in various disease states can be cultured in vitro using the same techniques as employed for peripheral cells, and these have also been found to have limited lifespans (eg. ref. 178). However, skin-infiltrating cells cultured in the presence of IL 4 in addition to IL 2 but in the absence of antigen presenting cells have been reported to grow apparently indefinitely. These cells were found not to harbour HTLV-1 (although it cannot be formally excluded that some other, thus far unidentified, virus is involved). These long-lived T cells manifested various chromosomal abnormalities at different frequencies (179). Generation of these lines was reproducible in different donors with different diseases, suggesting that isolation of apparently immortal cells under these conditions was not a rare event. While some of the donors were cancer patients, perhaps displaying generalized genetic instability and chromosomal fragility, the majority were atopic dermatitis patients (not known to fall into this category). Moreover, one T cell line was established from a skin nickel patch test which retained a normal karyotype up to 300 PD (way beyond the Hayflick limit) and acquired an abnormal karyotype thereafter (K. Kaltoft, personal communication, May, 1997). Therefore, depending on the source of cells and the culture conditions, normal T cells may be able to proliferate indefinitely, but the majority of evidence accumulated thus far suggests that this is the exception rather than the rule.