The main findings o f this study are: (1) Total C D 3 \ CD4^ and CD8^ T-cell subsets recover to within normal limits by 6 months post-HCT; (2) In looking for cell-surface markers which could define naïve, memory and effector T-cell subsets, it became apparent that CD45R A (when used in isolation) was not a reliable marker that would enable definition o f naïve CD8^ or CD4^ T-cells; (3) The majority o f peripheral blood T-cells displayed a memory or effector T-cell phenotype during the first 6 months post-transplant, with naïve T-cells appearing later than 6 months post-transplant; (4) Direct measurement o f thymic-dependent pathways revealed that the thymus does not contribute to T-cell reconstitution in the majority o f patients until after 6 months post-transplant; (5) The extent to which thymic-dependent T-cell recovery occurs varies between different individuals; (6) Reconstitution o f naïve T-cells is absolutely
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dependent on thymic output and does not occur without a concom itant increase in TREC levels.
The majority o f the haematological lineages recover relatively rapidly after HCT with the notable exception of the T-cell lineage, particularly CD4"^ T-cells, which have been reported to remain below normal healthy control numbers up to two years post-transplant (Behringer et a i , 1999; Isaacs, 2000; Lowdell et al., 1998; Small et al., 1999; Storek et al., 2001). In addition to the reduced numbers, distortions o f the T -cell compartment have also been reported in the early post-transplant period. A number o f reports have suggested that CDS"' T-cell recovery is relatively rapid, with slower recovery o f CD4"^ T- cells which results in a prolonged imbalance between helper (CD4^) and cytotoxic (CDS"") T-cell subsets (Atkinson et at., 1982; de Cast et al., 1985; Forman et a i , 1982; Janossy et a i , 1986; Mackall et al., 1997). Although distortions among individual T-cell subsets were evident in terms o f the recovery o f naïve, memory and effector T-cells, there was no imbalance in the recovery o f total CD4^ and CDS"^ T-cell subsets in this patient group as a whole. In the 26 HCT recipients studied, both CD4"^ and CD8^ T -cell subsets reached normal limits within 6 months after HCT.
However, several studies have shown that patients who restore normal T -cell numbers often remain susceptible to opportunistic infections and suggested that a defect in T -cell function may be one o f the reasons for such an increased susceptibility to infection. Further studies have revealed that restoration of T-cell function in patients after HCT is reliant on activation of thymic-dependent pathways (Dumont-Girard et a i , 1998; Roux et a i , 2000).
The second pathway of T -cell regeneration after HCT depends on the expansion o f mature, graft-derived T-cells and is termed the thym ic-independent pathway. Since this pathway is limited to the expansion o f a small number o f T -cell clones transferred with the stem -cell graft, this pathway restores T-cell numbers but does not generate a broad T- cell repertoire (Mackall et al., 1995; Mackall et al., 1993; Mackall and Gress, 1997; Rocha et al., 1989). The relative contribution o f thym ic-dependent and thym ic- independent pathways to T-cell reconstitution post-HCT has been studied using a variety o f methods as outlined previously.
In this study, the majority o f peripheral blood T-cells in the CD4 and CD8 com partment displayed a memory or effector cell phenotype between 3 and 6 months after HCT, suggesting that thym ic-independent pathways rapidly reconstituted the T -cell compartment after HCT. Such peripheral expansion o f memory and effector cells could be driven by a number o f processes including major or minor histocom patability differences between donor and recipient and/or viral antigens. Alternatively, this
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expansion could reflect normal homeostatic mechanisms that control the size o f the T -cell compartment.
A great deal o f work has been focused on understanding the regulation o f the peripheral T -cell pool, both under normal circumstances and under conditions o f severe lymphopenia. Several studies using mouse models have shown that naïve and memory T- cells, when transferred to a T-cell depleted host, have the ability to expand and proliferate to fill available space (B ell et al., 1987; Rocha et al., 1989; Tanchot et al., 1997). This so called homeostatic proliferation is likely to be very limited under normal conditions when lymphoid tissues contain large numbers of naïve and memory T-cells (Sprent et al., 1991). However, in patients given chemotherapy and/or pre-transplant conditioning, such as the 26 HCT recipients studied here, homeostatic proliferation o f naïve and m em ory cells via thymic-independent pathways could make up a large proportion o f repopulating cells in post-transplant patients. Historically, there has been som e debate on whether naïve T -cells convert to memory T-cells during the process o f homeostatic proliferation (Bell et al., 1987; Cho et a i , 2000; Goldrath et al., 2000; Rocha et al., 1989; Tanchot et al., 1997; Tough and Sprent, 1994). Recent studies suggest that naïve T -cells do convert to cells with a memory phenotype and do not revert back to a naïve phenotype on cessation o f proliferation (Cho et al., 2000; Tanchot et al., 2001); this point raises one of the limitations of this study. Previous studies have demonstrated that TREC are diluted out o f a cell population with each cell division (Douek et a i , 1998). The fact that TREC were not detected in the majority o f patients at 3 to 6 months post-HCT raises two possibilities. First and most obvious is that there was no thymic output in these patients. However, the second possibility reflects the ongoing processes of homeostatic proliferation, meaning that we cannot discount the possibility that naïve T-cells were exported from the thymus and stimulated to proliferate and differentiate into memory T-cells in response to hom eostatic signals. Thus it is feasible that thymic-dependent pathways were active at very early stages after transplant but that these pathways were being masked by the ongoing proliferative processes. In vivo experiments such as labelling o f thym ocytes with CFSE would be necessary to answer these questions.
In addition to these previous results, the identification o f non-naïve CD4^CD45RA^ T-cells in T -cell replete HCT recipients who do not experience rises in TREC levels raises questions on the role o f the thymus in regulating peripheral expansion. The inverse relationship observed between thymic output and non-naïve CD4^CD45RA^ T -cell numbers suggests that the thymus (or cells produced by the thymus) may inhibit the developm ent and maintenance o f this previously unidentified CD4"^ T -cell subset in patients after HCT. Equally, it is important to remember that although a statistical relationship exists in this instance, this does not necessarily represent a causal relationship since there are likely to be very com plex processes regulating thymic output and peripheral expansion in post-HCT patients. Nevertheless, previous studies analysing T -cell
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recovery in lym phopénie hosts have noted that thym ic-dependent pathways are used preferentially to peripheral expansion pathways in thymus-bearing m ice and that peripheral expansion is inhibited by the presence o f recent thymic emigrants (M ackall et al., 1996). The mechanism o f this inhibition remains unclear but may be related to the availability o f soluble factors such as IL-7 (Mackall et at., 2001). A second possibility is that the thymus produces cells that function as regulatory cells (such as the CD4'^CD25'’ subset) that inhibit homeostatic proliferation. “Bystander c e lls” that inhibit the extent o f homeostatic proliferation have been recently suggested to exist (Dumm er et al., 2 0 0 1 ). Together, these observations suggest that mechanisms exist to ensure that thym ic- dependent pathways are utilised to regenerate a diverse T-cell compartment whenever possible, but that peripheral expansion mechanisms restore T-cell numbers in the absence o f thymic output. Whether negative feedback signals are generated by an active thymus to inhibit peripheral expansion remains unknown.
The way in which these non-naïve CD4"^CD45RO'CD27' cells have evolved in vivo also remains unknown, but there are at least two potential mechanisms that could generate this phenotype. Previous studies have demonstrated that CD8^CD45RO C D 27‘ cells represent differentiated effector cells (Baars et a i , 2000; Hamann et al., 1997). Therefore, these cells may have evolved in a similar manner to differentiated CD8'^CD45RO CD27' effector T -cells and may represent so-called CD45RO ‘revertants’ . Secondly, it is possible that these cells represent naïve CD4"^CD45RO CD27"^ T-cells that have undergone hom eostatic proliferation and that the downregulation o f CD27 expression may represent a marker for homeostatic proliferation o f naïve CD4"^ T-cells in humans, similar to the upregulation o f C D 44 in the mouse (Tanchot and Rocha, 1995; von Boehmer and Hafen, 1993).
The homeostatic control o f T-cell numbers was discussed in Chapter 1. After HCT it is essential that normal homeostasis is restored for a functional immune system to be established. Several reports have made suggestions as to how this is achieved. Crucially, the restoration o f naïve T-cell numbers is dependent on thymic output, but recent thym ic emigrants may also have to com pete with resident naïve T-cells transferred with the stem cell graft. A previous study by Hebib et al. suggested that naïve (C D 45R A 0 and m em ory (CD45RO^) T -cells in patients during the first year after HCT were susceptible to spontaneous apoptosis when placed in short-term culture (Hebib et al., 1999). This study also demonstrated that the expanded memory cells were more susceptible to apoptosis than naïve cells. Hebib et al. further suggested that the increased susceptibility o f m em ory cells could be a mechanism to restore normal homeostasis. Thus, by their proposal, T -cells which exit the thymus would displace those cells which were most susceptible to apoptosis ie the memory cells. This is in contrast to the work o f Tanchot et al. who demonstrated that the naïve and memory T -cell compartments are independently regulated and that thymic output will only replace cells within the naïve compartment and not cells within the memory compartment (Tanchot and Rocha, 1995). Furthermore, the increases in naïve T-
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cell numbers in the 26 HCT recipients in our study were not accompanied by decreases in memory and effector T -cell numbers. However, it is unclear whether patients with low or absent thymic output will ever restore a phenotypically normal T -cell compartment, necessitating long-term analysis o f T-cell reconstitution to address this question. A recent study by Storek et al. (Storek et a i , 2001) analysed the T-cell compartment in long-term post-HCT survivors (20-30 years). They demonstrated that although CD4 TREC levels were significantly lower in HCT-recipients compared with normal individuals, this had no adverse consequences in terms o f their susceptibility to infection. The question remains therefore as to how important it is to restore a phenotypically “n orm al” T -cell compartment after HCT to be able to have adequate protective immunity.
While the measurement o f thym ic-independent pathways can be follow ed by reconstitution o f memory and effector T-cells, the measurement o f thym ic-dependent pathways after chemotherapy and HCT has been more problematic. The direct quantification o f thymic-dependent pathways has recently been enabled by the developm ent o f the TREC assay by a number o f different groups (D ouek et al., 1998; Okazaki and Sakano, 1988; Poulin et al., 1999). Using the assay published by D ouek et al. (D ouek et al., 2000), it was possible to directly measure the contribution o f thym ic- dependent pathways to T -cell reconstitution in HCT recipients. The results presented here demonstrate that thym ic-dependent pathways do contribute to T -cell reconstitution after HCT but that a number o f individuals have no detectable contribution o f the thymus to T- cell reconstitution. In addition, there was a lag-time o f between 6-9 months before thym ic out put could be detected in the majority o f patients. This delay in thymic output is significantly longer than in patients who received chemotherapy (Mackall et al., 1995), but has been observed in previous studies o f allogeneic HCT recipients (Roux et al., 2000). These observations suggest that som e time is necessary to reorganise thym ic- architecture and education o f thym ocytes from T -cell precursors.
In conclusion, this work has shown that both thym ic-dependent and thym ic-independent pathways are active in allogeneic HCT recipients. Furthermore, these results demonstrate that it is essential that both pathways contribute to T -cell reconstitution after HCT. In the context o f T -cell reconstitution after HCT, thym ic-independent pathways restore T -cell numbers, but not necessarily fully competent T -cell mediated immunity in the first six months after HCT. After six months post-HCT, thym ic-dependent pathways can be detected and begin to restore naïve T-cell numbers and the diversity o f the TCR repertoire.
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