SÍNTESIS Y EVALUACIÓN DE RESULTADOS

The previous results demonstrate that the regeneration o f peripheral CD4"^ T -cell numbers post-HCT is reliant on thym ic-independent pathways in the short term with thym ic- dependent pathways contributing to CD4^ recovery in the longer term. The reconstitution o f naïve CD8^ T-cells has been studied to a lesser extent than CD4^ T -cells due to the difficulties associated with identification o f naïve CD8"^ T-cells. This is highlighted again in this patient cohort through the analysis o f CD8"^ T-cells with a CD45RA^ phenotype (Figure 3.6A ). At three months post-HCT, the median number o f CD8^CD45RA^ T-cells was 46 cells per pi (Range 0 to 1950). At 9 months post-HCT 11 o f 16 patients had restored CD8^CD45RA^ T-cell numbers within the normal range with median

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CD8XD45RA'" T-cell numbers of 154 (range 0-861). However, it is clear from the data presented previously that there is more than one species o f T-cell contained within the CD8 CD45RA^ subset and that CD45RA does not provide a reliable marker on its own to define naïve CD8^ T-cells.

3000 1 0 0 0- IO O -- lO-l M o n th s p o st-tra n sp la n t

B

D

Figure 3.6: R ecoveiy o f naïve, memory and effector CDS'" T-cell subsets

A b s o lu te n u m b e rs o f C D 4 5 R A T O (A ), n a ïv e C D 4 5 R 0 C D 2 7 " (B ), n o n -n a ïv e (C ), an d C D 5 7 T D 2 8 (D) C D S "

T -c e lls w e re c a lc u la te d fro m a b s o lu te n u m b e rs o f CDS'" T -c e lls as d e s c rib e d p r e v io u s ly . T h e g re y d a s h e d l i n e s

in (A ) r e p re se n t th e u p p e r an d lo w e r lim its o f C D 8 X D 4 3 R A " T -c e ll n u m b e rs in n o rm a l in d iv id u a ls ( G o d th e lp

e t a i , 1999). R e fe re n c e v a lu e s fo r F ig u re s (B ), (C ) an d (D ) w e re n o t d e te rm in e d .

The recovery of the truly naïve CD8^CD45RO CD27^ subset (Figure 3.6B) was slower than that o f the CD8^CD45RA'' subset. At 3 months post-HCT, the median number o f C D 8 T D 4 5 R O C D 2 T ‘ T-cells was 2 cells per pi (Range 0 to 45). Although this subset increased with time post-transplant the median number of CD8"CD45ROCD27^ T-cells at 12 months post-transplant remained low with a median o f 76 cells per pi (Range 0 to 541). A number of previous studies have used only CD45RA to define naïve CD8^ T-cells, but it is clear from these results that this would result in a substantial overestimate o f naïve circulating CD8" T-cell numbers in post-HCT patients.

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The recovery o f non-naïve CD8"^ T-cells was rapid (Figure 3.6C) with the median num ber o f non-naïve CDS'" T-cells being 179 cells per pi (range 0 -36 99 ) at 3 months, rising to a median o f 307 cells per pi (0-2497) by 12 months post-transplant. A number o f patients restored very high non-naive CDS^ T-cell numbers that were significantly higher than the reference values for the total CDS compartment. This has been observed in a number o f previous studies although the exact reason for this CDS ‘overshoot’ is unclear (D um ont- Girard et al., 199S; Singer et at., 19S3; Witherspoon et at., 19S2).

As mentioned previously, due to the heterogeneous expression o f CD45RO and CD27 on different CDS^ T-cells, it was difficult to distinguish discrete populations o f memory and effector CDS^ T-cells in the majority o f patients. I therefore looked at the absolute number of CDS^ T-cells which were C D 57’^CD28 . These cells represent CDS^ T -cells that have undergone extensive cell division and can therefore be used as a marker for thym ic- independent T -cell reconstitution. Similar to the total CDS’" T-cell population and the non-naïve CDS"^ T-cell population, there were significant numbers o f CD8^CD57’^CD2S cells at 3 months post transplant (Figure 3.6D ) with a median number of 77 cells per pi (range: 0-2683). The median number o f C D 8’^CD57"^CD28‘ cells increased to 190 cells per pi (range: 0 -2 24 4) at 12 months post-transplant. The normal range o f CD8 T -cell counts is between 200-900 CD8^ T-cells per pi, with naïve T-cells making up the bulk o f the CD8 population in healthy controls. The C D 8’^CD57’^CD28‘ subset forms only a m inor subset in normal individuals but can make up the majority o f T-cells in patients after HCT. This observation, along with previous reports on the evolution o f C D 8’’C D 57’^CD28 T-cells suggests that the short term reconstitution o f CD8" T -cell numbers occurs primarily through thymic-independent pathways.

Table 3.3: Summary o f reconstitution o f naïve, memory and effector C D 8^ T-cell subsets

M onths Total CD8* N aïve CD8* N o n -n aiv e CD8* 008*57*28 p o s t-tra n s p la n t 3 180 (4-3699) 2 (0-45) 179 (0-3699) 77 (0-2683) 6 350 (5-3057) 17 (0-74 246 (0-3057) 161 (0-1410) 9 3 3 0 (1 0 -2 0 9 3 ) 1 5(0-136) 209 (0-2093) 104 (0-1562) 12 5 2 0 (1 0 -2 4 9 7 ) 76 (0-541) 307 (0-2497) 190 (0-2244) A ll v a lu e s a re g iv e n a s c e lls p e r m ic r o litr e o f b lo o d . T h e m e d ia n n u m b e r s o f e a c h CDS^^ T -c e ll s u b s e t w e re

c a lc u la te d f o r th e 2 6 p a tie n ts a s d e s c rib e d p re v io u s ly . M e d ia n v a lu e s a re s h o w n w ith th e r a n g e o f e a c h s u b s e t

in b r a c k e ts .

Similar to the results from the analysis o f the reconstitution o f CD4^ T -cell subsets, the recovery o f C D 8’' T-cells occured in the short term by expansion o f CD8'' T -cells with a memory or effector cell phenotype (Table 3.3). Therefore, thym ic-independent pathways were able to rapidly restore C D 8’^ T-cell numbers to within the normal range between 3 -6

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months post-HCT. The recovery o f naïve CD8^ T-cells was slower and remained low even at 12 months post-HCT in some patients. A lso similar to the CD4"^ T-cell subset, there were a number o f patients with low but detectable numbers o f naïve CDS'” T -cells at 3 months post-transplant.

Normality testing of the CD8^ subpopulations revealed abnormally distributed populations within the CD45RA^ subset at 3, 6 and 9 months post-HCT (P = 0.0004, P = 0.04 and P = 0.0003 respectively). The CD8"^CD45R0CD27^, non-naive C D 8‘" and CD8^CD57"^CD28' subsets were all abnormally distributed at 3 months post-HCT (P = 0.0014, P = 0.028 and P = 0.032 respectively), but were restored to a Gaussian distribution after 3 months post-HCT. The suggestions from this data are two-fold; first, similar to the CD3^ and CD4^ populations, factors associated with HCT may affect the repopulation o f the T-cell pool; second, since all CD8 subpopulations are affected at 3 months post-HCT, CD8"^ T-cells may be more susceptible to factors that stimulate or prevent proliferation o f cell death compared with the CD4"’ T -cell population.

The previous results demonstrate that both CD4"^ and CD8"^ T -cell numbers are restored in the short term (less than 6 months post-HCT) through the expansion o f T -cells with a memory or effector phenotype, while in the longer term, naïve T -cells increase in number.