1.7. ASPECTOS FUNDAMENTALES QUE TIENEN MAYOR IMPACTO
1.7.2. ÁNGULO DE FASE
Previous studies have shown that the expanded CD8+ T cell pool that is present during AIM is susceptible to growth factor deprivation-induced apoptosis. The next investigation was carried out in order to determine whether this was also true for EBV- specific CD8+ T cells within the total CD8+ pool of patients with AIM (Fig. 5.3.3.). When cultured in the absence of exogenous growth factors, the total CD8+ T cells from patients with AIM perish rapidly by apoptosis resulting in <20% and <5% survival after 1 and 5 days of culture respectively (Fig. 5.3.3.A.). During AIM, both lytic and latent epitope-specific CD8+ T cells were equally susceptible to rapid apoptosis (only data for lytic epitope-specific cells is shown; Fig. 5.3.1.A.). W hen cells from individuals that have recovered from AIM were cultured under identical conditions, it was found that the total CD8+ T cell population was now relatively resistant to apoptosis after 5 days of culture (Fig. 5.3.3.B.); (65% survival after 5 days of culture). EBV epitope-specific CD8+ T cells that were present after AIM were more susceptible to death than the total CD8+ population (35% vs 65% survival after 5 days of culture) but more resistant to death when compared to lytic epitope-specific CD8+ T cells that are found during AIM (35% vs 5% survival after 5 days; p<0.01). The enhanced survival of the total CD8+ T in individuals after recovery from AIM relative to virus-specific populations was probably due in part to the presence of apoptosis-resistant naïve CD8+ T cells in the former but not the latter population.
The relative survival in culture of EBV-specific CD8+ T cells that do or do not re-express CD45RA (Fig. 5.3.3.C.) was determined next. In these experiments only lytic epitope-specific populations were investigated since there were generally too few CD45RA re-expressing latent virus epitope-specific cells to investigate accurately (Fig. 5.3.1.). It was found that in each of 5 experiments performed that EBV-specific cells that re-express CD45RA showed better survival than the cells that did not express this molecule (60.8 ± 15.3 vs 30.2 ± 11.7 respectively; p<0.012).
Chapter 5 -S 2(r 0 1 3 5 Days in Culture
B
^ 0 0 ] ^ 8 0 'I
5
401
=S 20" u 0 0 3 5 7 Days iu Culture ^lOC 80 > 60 I 40 = 20 U 0^ RAK RAK RAK GLC GLC CD45RA* CD45RA Tetram er gatedFigure 5.3.3. Susceptibility of virus-specific CD8+ T cells to apoptosis. PBMC were isolated from donors with AIM and cultured in the absence of exogenous mediators (A). The total viable cell recovery was measured at various times after culture. In addition, the percentage of total CD8+ T cells or virus-specific (RAK lytic epitope) CD8+ T cells was also determined in the cultured PBMC populations. This enabled the absolute number of total CD8+ T cells and EBV-specific CD8+ T cells to be determined after culture. The survival of the cells after culture was expressed as a percentage of the original number of cells at day 0. The survival o f total CD8+ and EBV-specific CD8+T cells in culture was also determined in patients who had recovered from AIM (B). The absolute number of CD45RA+ or CD45RA- virus-specific CD8+ T cells at different times after culture was determined by multiplying the percentage of these cells that were present at different times by the cell recovery (C). The percent survival of CD45RA+ or CD45RA-EBV-specific CD8+ T cells was determined relative to the initial input at day 0. The results shown in (A) and (B) are the mean and S.E.M. of triplicate determinations for the survival of cells from 1 individual each and are representative of at least 4 separate experiments that have been performed in each case. In (C), the relative survival of EBV-specific CD8+ T cells, which do or do not re express CD45RA in 5 different individuals that have been tested, are shown.
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5.3.4 IL-2, IL-15 and FCM can rescue CD3+CD8+ T cells and EBV
RAK-specific CD8+ T cells from CDD
PBMC obtained from healthy donors who had recovered from AIM at least 10 years previously were placed in culture in the absence or presence o f survival factors and viable cell recovery percentages were determined 7 days later. IL-2 and IL-15 induced proliferation in both total CD8+ T cells and in antigen-specific T cells thereby increasing cell recovery (Fig. 5.3.4.). FCM did not induce proliferation but did partially rescue total CD8+ and antigen-specific T cells from apoptosis (Fig. 5.3.4.). Viable cell recovery percentages differed significantly from control conditions which T cells were incubated in medium alone (p<0.01).
250 - ■ Total CD8+ □ Total TET+ P 150
^
100 50control IL-2 IL-15 FCM
Figure 5.3.4. IL-2, IL-15 and FCM can rescue CD3+CD8+ T cells and EBV RAK- specific CD8+ T cells from CDD. Freshly isolated PBMC from donors who had recovered from AIM were placed in culture for 7 days in the absence or presence o f 20 units/ml o f IL-2, 20 units/ml o f IL-15 or 50% FCM. PBMC were stained with mouse CD3, CD8 and MHC class I tetramer specific for RAK peptide conjugated to phycoerytherin (PE) on day 0 prior to culture and 7 days later. The percentage of CD3+CD8+ T cells was determined from the viable forward by side scatter gate whereas the percentage o f CD8+tetramer+ T cells was determined from the CD3+CD8+ gate. VCR% were calculated based upon cell number added to each well on day 0. These results are representative of 3 separate experiments.
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