Derivaciones de la historia y visiones de la pedagogía

An important factor in the long-term recovery of the patient after high dose chemotherapy and BMT is that the immune system is reconstituted and a competent immune repertoire is re-established. Numerical recovery of the components of the bone marrow is just one aspect; functional recovery of the cellular interactions of those components is also vital. Various factors may affect how well the immune system recovers. The ablation of the host immune system during conditioning, the manipulation of the donor graft, the sustained transfer of donor-antigen specific immunity and the age of the patient are all significant.

It has traditionally been accepted that the patient’s ability to recover a fully diverse functioning immune system depends on age, and the number of T cells returned to the patient in the graft. A child has a functioning thymus, but with age, post-BMT immunosuppression and GvHD the ability of the thymus to function diminishes (reviewed in Parkman and Weinberg, 1997). Recently it has been proposed that a relatively high degree of thymic activity does survive into adult life. It is suggested that through late middle-age adults retain thymic activity though it would be several log folds lower than a child (Douek et al.,

1998). Therefore a relatively diverse immune repertoire may eventually be attainable, in the post-BMT patient.

The possibility may also exist for adults to expand their immune repertoire by extra-thymic T-cell differentiation. The gut mucosa and liver have been suggested as possible sites of T-cell differentiation (Rocha et al., 1995) although other tissues, including skin, may be shown to be important.

A major complication in BMT is the risk of life-threatening infections occurring in the weeks post-BMT. A naïve or dysfunctional immune system resulting from chemotherapy and BMT conditioning leaves the patient at risk of bacterial, fungal and viral, infections which can be fatal (Kook et a/., 1996, Parkman and Weinberg, 1997, Kruger et a/., 1999). BMT can produce prolonged, profound and rapid neutropenia, an ideal environment for bacterial infections such as Pseudomonas spp. to flourish. The advent of recombinant GCSF (Granulocyte colony stimulatory factor) has reduced the average period of post-transplant neutropenia to less than 15 days (the median time to 0.5x1 O^neutrophils/I is over 20 days (Hale et a/., 1998)).

Fungal infections may also be a major cause of mortality. In a recent report Aspergillus spp. and Candida spp. accounted for over half of all fatal infections (Kruger et a/., 1999). As resistance to fungal infections is chiefly mediated by T cell immunity T cell depletion can reduce the body's ability to control fungal infections (Klein and Horejsi, 1997).

The lack of antigen-specific T lymphocytes also leaves the body vulnerable to viral infections (reviewed in Parkman and Weinberg, 1997). One of the most

There are a number of ways in which a transplant recipient can be infected. A seronegative bone marrow transplant patient may be infected by blood products, or by the marrow itself. If the patient was already seropositive they may be re-infected by another strain of CMV from blood products or marrow, or by their own endogenous strain of CMV which was latent but becomes reactivated in the immunocompromised patient (Prentice et a/., 1998). Other viral infections that can result in serious complications include hepatitis B, hepatitis C and Epstein-Barr virus (EBV). The latter is of particular importance in patients who receive TCD bone marrow transplants (Westmoreland, 1998).

Investigators have noted the rapid recovery of NK cells post-BMT (Reittie et a/., 1989; Lowdell et al., 1997). These are largely "true" NK cells rather than NKT cells, particularly in patients such as those studied here who have received a T cell depleted donor graft. They may have an important role, possibly fulfilling the function of T cells prior to their recovery. Functional tests have shown that NK cells recover their activity not only against K562 (a traditional NK target) but also are active against T and B cell targets (HSB2 and Daudi or EBV- transformed B cells respectively)(Rooney et a/., 1986). This can occur as early as 4-6 weeks after transplantation, during the period when T cells are recovering slowly, particularly after a TCD BMT.

It is known that NK cells from normal donors can react to T and B cell targets if given the appropriate stimulation {in vitro, on exposure of NK cells to IFN-y). In BMT patients high levels of IFN-y have been identified in the body soon after bone marrow transplant (Antin et al., 1992). In this group of BMT patients NK

cells were active against T and B cell targets without the possible side effects of T cell activity which could include GvHD, CMV or apparent graft rejection (Rooney etal., 1986).

NK cells have also been shown to play an important role against certain strains of CMV. One group have demonstrated that cell surface LFA-3 (lymphocyte function-associated antigen-3) expression is crucial to sensitivity or resistance to NK cell lysis. This group used fibroblasts infected with different stains of CMV, and those that up-regulated LFA-3 were susceptible to lysis and those that down-regulated LFA-3 were resistant. This change in LFA-3 expression was mediated by CMV immediate early or early CMV genes (Fletcher et a/.,

1999).

The type of graft used (allogeneic or autologous) can present the immune system with different problems. Prior to the use of peripheral blood stem cell transplants (PBSCT), transplants were performed either with autologous bone marrow taken from the patient in CR, or with bone marrow from an HLA- identical sibling or volunteer unrelated donor. PBSC transplants have become increasingly popular, and this form of transplant may determine a different outcome for the immune system of the patient than BMT due to larger numbers of T cells in the graft and the possible effects of GCSF used in the mobilisation of stem cells (Tayebi et a/., 2001, Volpi et a/., 2001). In the studies presented here all transplanted patients received bone marrow.

targets the thymus and dendritic cells in the skin, liver and spleen and leads to severe reduction in the rate of reconstitution of CD4^ T cells. In contrast, TCD for the prevention of GvHD leads to reduction in the T cell pool from which peripheral T cell expansion can occur - also leading to delayed T cell restoration. Treatment for severe GvHD may have similar effects.

With an autologous transplant the conditioning regimen is designed to eliminate residual tumour load. The immune system does not require complete ablation because the incoming graft is clearly seen as ‘self and therefore there is no risk of rejection. Similarly TCD is not required since there is no risk of GvHD. This also obviates the need for immunosuppressive drugs, which may interfere with immune recovery. The autologous setting therefore provides an opportunity to study the re-emergence of the immune system after chemotherapy and radiotherapy without other factors obscuring the picture (although all transplant recipients receive prophylactic treatment against infections, which may also have a bearing on immune recovery).

In this study patients receiving four different types of bone marrow transplant; manipulated, partially manipulated and unmanipulated allogeneic transplants and autologous transplants were followed up. A panel of antibodies (as detailed in Table 2.1) was used to study lymphocyte differentiation antigens to give an indication of the dynamics of immune system recovery, and the effects of TCD. CD4 and CD8 are surface molecules expressed on subsets of T cells and CD45RA and CD45RO were used to identify naïve and mature cells respectively and appear in conjunction with both CD4 and CD8.

The CD56 antigen is expressed on 95% of natural killer cells. CD56 and CD16 are the most commonly used and most informative molecules denoting NK cells with over 95% of such cells expressing CD56 (Robertson and Ritz, 1990). A small proportion of NK cells in normal donors lack CD56 but express GDI6. In preliminary studies this subset CD567CD16^ represented <2% of CD37CD19‘ cells in normal donors and transplant recipients and so CD56 alone was used. Data from previous studies in the department (Lowdell et al., 1995) had shown that NKT cells were uncommon in transplant recipients which further supported the use of CD56 alone as a molecule denoting NK cells. The analysis of the CD8"' subset of NK cells was aided by the fact that CD56VCD3' NK cells express only the CD8a chain as a homodimer and appear as CD56VCD8'^'^^ cells when analysed by flow cytometry. In contrast, the subset of NKT cells which expresses CD8 (approximately 60%) shows levels of CD8 which are equivalent to T cells and also co-express CD57 (Ortaldo et a!., 1991).

A recent study into individuals that had received TCD-marrow from HLA- identical siblings (TCD by Campath 1G) found that there was a significantly reduced mean lymphocyte count of cells expressing CD3 at 8 weeks post transplant (p=0.05), but that populations expressing CD8 and CD56 remained within the normal range throughout the study. Normalisation of cell numbers of CD3^ cells did not take place until 52 weeks post-transplant (Davison et a/., 2000). Also a study from 1997 into repopulation of circulating T, B and NK cells post autologous and allogeneic BMT and PBSCT without TCD, found that whilst

in all groups, the CD56VCD3^ subsets did not recover until between 9-12 months post-transplant (Parrado et al., 1997).

HLA-DR was used to denote activation of T cells and CD28 is the ligand for CD80 required for costimulation of T cells.