Telomerase Activity in Virus Specific CD8^ T Cells During Acute Infection

Telomeres are synthesized by telomerase, which can be up-regulated following T cell activation, thus counteracting telomere shortening. In order to investigate whether the telomere length preservation seen in cells from AIM patients was related to increased telomerase activity, the relevant cell subpopulations from AIM patients PBMC were isolated and analysed for telomerase activity. CDS^ T cells from AIM patients were isolated by negative selection. This fraction was then enriched for tetramer binding cells by positive selection (see methods chapter). The CD8^, tetramer-enriched and the CDS-depleted cell fractions were all analysed for telomerase activity and the results are shown in Figure 51. Both the CD8^ and the tetramer-enriched fractions showed high telomerase activity during the acute phase of infection. Lower but

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detectable levels of telomerase activity was found in the CD8 depleted subset confirming previous observations [508] showing that telomerase is activated in CD4^ T cells in AIM.

Telomerase activity was then investigated in patients after resolution of the infection. Telomerase activity was measured in isolated CD8^ and tetramer enriched cells from the same patient after resolution of AIM. It was found that upon follow up, there is a marked reduction of telomerase activity in both the CD8^ and tetramer positive cells compared to the same cells in AIM (Figure 51). There is however low but detectable telomerase activity in both these subsets suggesting a residual level of activation in these cells. In five patients studies to date, CD8^ T cells that were isolated during AIM all had high telomerase activity relative to CD8^ T cells from the same patient after follow up (data not shown). This suggests that the ability to maintain telomeres of EBV-specific CD8^ T cells is due to the ability to up regulate telomerase during the acute phase of disease. However, since telomerase activity decreases after AIM, telomere maintenance or shortening of EBV-specific CD8^ T cells will depend on the capacity of these cells to up regulate telomerase upon subsequent challenge with virus after longer periods in vivo. Longitudinal studies are now in progress to address this point.

6. Flow-FISH in Antigen Specific CD8^ T Lymphocytes 144 AIM Follow-up

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Figure 51 EBV-specific CD8^ cells expresses high telomerase activity during AIM. CDS T cells were first isolated from a PBMC population from AIM patient. B8-RAK tetramer positive cells were then isolated from the CDS population using anti-PE MACS beads. Telomerase activity was determined using the TRAP assay autoradiography performed on C D S \ CDS' and tetramer enriched T cell fractions from the patient during AIM and 12 months after follow-up (A). The TRAP-eze protocol, in which telomerase activity is amplified by PCR, gives rise to a ladder of products with 6 base pair increments, starting at 50 nucleotides. Heat inactivated (HI) cell extracts provided a telomerase negative control for each sample. The negative control (-CNT) contains the PCR mix without the cell extract. In addition, amplification of the internal control oligonucleotides together with the TS primer give rise to the internal PCR control (36 bp). Telomerase activity in acute and follow-up IM patient activity was expressed as total product generated calculated according to manufacturer’s instructions (B). Each Unit of total product generated corresponds to the number of TS primers extended with at least 4 telomeric repeats by telomerase in the extract in a 10 minute incubation at 30®C. Black bars represent telomerase activity in acute IM patients while open bars refer to follow-up patient samples. In a total of 4 patients studied, high telomerase activity was found in CDS T cells during AIM, which was substantially reduced in the follow-up samples which were taken after 12 months or longer after resolution of the acute infection

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6.6 Discussion

In this chapter, the new technique for measuring telomere length by flow cytometry described in the previous chapter, was used to investigate replicative senescence in antigen specific CD8^ T lymphocytes during and following immune responses. Infection by EBV was used as model due to its induction of high levels of lymphocyte proliferation during the acute stage of disease and for accessibility to patients at various stages of disease. Acute infection by EBV is characterised by large clonal expansions of antigen specific CD8^ T lymphocytes that kill infected cells. It was found that during the acute stage of disease, when large clonal expansions of highly activated lymphocytes populate the peripheral blood and lymphatic organs, EBV specific CD8^ T lymphocytes maintain their telomere length as compared to CD8^ T lymphocytes from normal individuals and also compared to their own CD4^ T cells. However, the results shown also suggest that the CD4^ T cells pool in these patients also have increased telomere length suggesting that they have also been activated and have a role in the acute phase of this infection. We only analysed cells within the non- blastoid gate which excludes the possibility that any differences between the subsets analysed arose from the increased FISH signal resulting from telomere replication in cycling cells. Although the cells from AIM patients which fell in this gate were not blasts, the majority expressed HLA-DR suggesting that they had been previously activated and were likely to be recently activated post-mitotic cells rather than resting cells. The increased telomere length of CD8^ relative to CD4^ T cells in AIM patients detected by Flow-FISH analysis confirms previous reports [508] using DNA analysis by Southern blot. The maintenance of telomere length of EBV-specific CD8^ T cells in AIM is associated with induction of telomerase activity and suggests that the CD8^ T cells which escape apoptosis during resolution of AIM, enter the memory pool with a relatively preserved replicative capacity.

In order to investigate this ftirther, samples were collected from patients at several times following recovery from AIM. These were investigated for telomere length in antigen specific lymphocytes during and following AIM.

It was found that in 3 out of 4 individuals, telomere length remains virtually identical up to 1 year after disease resolution. In patients that were studied for longer periods after AIM, there was consistent telomere shortening. The ability to maintain telomere length is not related to the capacity of different individuals to upregulate telomerase

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activity in the acute phase of disease since all the patients studied during AIM have upregulated telomerase activity in the CD8^ T cell population.

Telomere shortening at latter time points following disease resolution may be explained by the nature of EBV infection itself which, unlike other viruses, causes persistent infection. The persistence of the virus in the host results in episodes of viral replication, leading to re-activation of memory CD8^ T lymphocytes. It is not known to what extent re-activation takes place or, more importantly, to what extent telomerase is up-regulated during these episodes. It has been described however, that telomerase loses its ability to be up-regulated upon repeated stimulations in vitro. This suggests that depending on the frequency and nature of the re activation episodes in a given host and the ability of telomerase to be increased concomitantly with proliferation, so telomere shortening may vary between clone and some may become senescent and eventually disappear. This is indirectly supported by findings showing that the acute stage of disease is dominated by a few highly expanded clones. These clones are not always found in memory and, probably due to continuous viral challenge, are replaced by others.

The decreased ability to increase telomerase upon repeated stimulations, which could be the basis for telomere shortening and possibly senescence of certain clones following EBV infection is an issue that requires further investigation. It has been shown for example, that optimal telomerase induction requires CD28 ligation and at the same time it is known that this molecule is down-regulated after activation in human CD8^ T lymphocytes. A proportion of CD8^ T lymphocytes in AIM have lost CD28 [15] and CD28'CD8^ T cells have been described to have shorter telomeres than their CD28^ counterparts [350]. This suggests that either telomerase cannot be significantly increased in memory responses or that other co-stimulatory molecules or cytokines may overcome this pathway. Further studies are required to clarify these issues. However, the two colour Flow-FISH technology described here will enable further detailed investigation of the heterogeneity of telomere length changes in patients at multiple time points after acute infection. This information is clearly crucial for the clarification of the role of replicative senescence in the regulation of CD8^ T cell memory.

In conclusion, the use of MHC class-I tetramer technology together with the ability to analyse changes in telomere length in these cells by two colour Flow-FISH now

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allows the study of replicative senescence in memory CD8^ T cells in humans. This will allow the investigation of changes within cells that are reactive to different epitopes of viruses in response to vaccination and will clarify if replicative senescence may be the reason why immunity to some vaccines offer life-long protection while others are only effective for a limited period. This technology will also enable the investigation of antigen-specific CD8^ T cell responses in other viral infections such HIV, and will enable studies into the reasons for the compromised antigen specific T cell responses in the elderly.

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