4.1 Introduction
4.1.1 R econstitution o f yc chain expression in patients follow ing treatm ent by BMT
In the m ajority o f XSCID patients analysed in the previous chapter, the nature o f the disease causing m utation abrogated stable yc chain expression. In these cases it was
possible to analyse the reconstitution o f yc chain expression following treatm ent by BM T, as a tool in m onitoring the levels o f donor bone m arrow engraftm ent. The four patients analysed and the type o f BM T treatm ent they received are indicated in Table 4.1. D onor cells were distinguishable by the presence o f yc chain expression on the cell surface. Im m unofluorescent staining with the yc chain antibody in conjunction with T
and B lym phocyte and m onocyte lineage specific markers, enabled the levels o f yc chain expression w ithin the different haem atopoietic lineages to be determ ined. The aim o f this study was to determ ine the different levels o f donorirecipient chim aerism within the different lineages and gain some inform ation about the kinetics o f engraftm ent o f the different cell types.
Table 4.1: Bone m arrow transplantation conditions o f patients analysed in this
study
Patient Bone m arrow tran splantation con d itions DC Unrelated, HLA-matched BMT, with conditioning
OV Unrelated HLA-matched T cell depleted BMT, with conditioning AKAD HLA-matched sibling infusion, without conditioning
JA H ap loid en tical, T cell depleted maternal BMT, with conditioning, CD34 enhanced PBSC infusion
4.1.2 C linical m onitoring o f bone marrow engraftm ent follow ing BM T
Bone m arrow engraftm ent is monitored clinically by fluorescence in situ hybridisation (FISH) analysis and restriction fragm ent length polym orphism (RFLP) (Leclair et al., 1995; van Toi et a l , 1998). In the discussion, com parison is made betw een chimaerism data obtained by FISH and RFLP analyses perform ed by the Bone M arrow Laboratory, D epartm ent o f H aem atology, Great Ormond Street Hospital for Children NHS Trust, London, and the yc chain expression data presented in this study.
FISH is a m olecular cytogenetic technique involving the hybridisation o f a fluorescently labelled single-stranded DNA probe to a m etaphase chrom osom e spread. A metaphase spread contains the intact denatured chromosom al DNA o f cells, on a microscope slide, allow ing separate chrom osom es to be distinguished by direct visualisation o f a fluorescent signal produced by hybridisation o f the probe to the relevant chromosome. Engraftm ent in sex-m ism atched BM T is monitored routinely by FISH analysis o f m ononuclear cells and neutrophils, as female (XX) and male (XY) cells can be differentiated by the utilisation o f fluorescent probes that recognise repetitive elements on the X and Y chrom osom es. Simultaneous usage o f X and Y chrom osom e specific probes, as opposed to ju st one sex chromosom e probe, pushes the levels o f detection in 2 colour FISH to >98% for XY and >96.5% for XX (van Toi et a l , 1998).
Restriction fragm ent length polym orphism (RFLP) is a DNA based technique that is used in the assessm ent o f the degree o f engraftm ent following BM T as well as to track disease genes through families (Blazar et a l , 1985). Sequence variations or polym orphism s, such as highly polym orphic m inisatellite regions o f DNA which consist o f variable num bers o f tandem repetitive units in non-coding DNA, are frequent tliroughout the genome. I f they affect restriction endonuclease recognition sites, DNA fragments o f different sizes will be produced following restriction enzym e digestion o f the DNA. The different allelles produced are called restriction fragment length polym orphism s (RFLPs). The genom ic fragments are separated by electrophoresis, transferred by Southern blotting to a m em brane and hybridised w ith a labelled probe. D ifferent individuals exhibit distinct and specific hybridisation patterns or RFLPs, enabling donor and recipient cells to be distinguished. RFLP is less sensitive than FISH due to lim iting technical factors with the capability o f detecting a m inor population o f cells constituting 10% o f the total num ber (Blazar et al., 1985).
4.2 Results
D ifferent levels o f donorirecipient chim aerism are apparent in the separate haem atopoietic lineages. T-lym phocytes in all cases and m onocytes in three out o f the four patients, exhibited jc chain expression com parable to norm al controls whereas
reduced levels o f yc chain expression on the B-lym phocytes w ere suggestive o f mixed chim aerism . The results are sum m arised in Table 4.2.
Table 4.2: Sum m ary o f levels o f yc chain expression on patient and norm al cells
Patient T im e point post- BM T
P ercen tage o f patients cells exp ressin g y,. chain (w ith n orm al con trol levels show n in brackets) T -lym ph ocytes B -lym phocytes M onocytes
DC 7weeks 100 (1 0 0 ) 21 (95) 100 (100)
DC 12weeks 9 9 (1 0 0 ) 67 (93) 9 5 ( 9 9 )
OV Syears 100 (100) 7 8 (8 9 ) N D
AK A D 3 years 91 (89) 1 3 (5 3 ) 11 (56)
JA 16months 97 (100) 73 (93) 97 (99)
A nalysis o f both patients DC and JA dem onstrated full yc chain expressing donor cell engraftm ent o f T lym phocytes and m onocytes, due to normal levels o f expression, with reduced expression observed on B cells. O nly 73% o f patient JA ’s B lymphocytes expressed protein in com parison to 93% in the control, suggesting that some o f the patients ow n yc chain deficient cells remain (Fig 4.1). Interestingly, im m unostaining o f patient D C ’s B cells revealed a significantly lower level o f yc chain expression at 7weeks, 21% com pared to 95% in the control, w hich increased to 67% 5weeks later (Fig 4.2). These results indicate that the donor B lym phocytes engrafted at a lower level and at a slow er rate than the other two lineages.
N orm al levels o f yc chain expression by T lym phocytes and slightly reduced expression levels by B lym phocytes were revealed follow ing im m unostaining o f patient OV cells three years after BM T treatment, yc chain expression on the patient’s B lymphocytes was only partially reduced from normal w ith 78% o f cells expressing as compared to 89% in norm al control (Fig 4.3). Patient OV appears to be im m unologically normal w ith full engraftm ent in the T-lym phocyte lineage in addition to high levels o f donor
Fig 4.1: Yc chain expression on PB derived T lymphocytes, B lymphocytes and monocytes from patient JA 16months post BMT and a normal control.
Patient JA Control - 93% . . 73% CD 19 expression Fluorescence intensity
(Yc chain expression) lo^
lOU
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I r
CD3 expression