MERCADO FORWARD

--- CD 14 expression ---►

T able 4.3: Percentage o f cells o f donor origin analysed by FISH and RFLP

Patient C ell type Percentage o f d onor cells T ech niqu e

A K A D W hole blood leukocytes 50 RFLP

OV BM leukocytes 60 RFLP

JA Mononuclear cells 84.4% FISH

DC Mononuclear cells 98.6% FISH

DC Mononuclear cells 97.5% FISH

RFLP and FISH analyses w ere perform ed by the Bone M arrow laboratory, Departm ent o f H aem atology, G reat Orm ond Street Hospital for Children N HS Trust, London, on the same days as the yc chain expression studies referred to in the text. RFLP analysis com prised the Hin fl restriction endonuclease digestion o f patient high m olecular w eight D NA, Southern blotting and hybridisation w ith a non-isotopic chem ilum inescent single locus probe (NICE^^ probes, Cellm ark D iagnostics). The percentage o f donor cells was quantitated by gel scanning techniques w hen partial engraftm ent was noted by the appearance o f m ixed donor and host genotype pattern. Chim aerism after sex- m ism atched BM T treatm ent was investigated by fluorescent in situ hybridisation (FISH) analysis using the CEP® X SpectrumOrange'^^/ Y SpectrumGreen^^^ direct labelled fluorescent DNA probe kit (Vysis, Inc.). M etaphase spreads were prepared from unstim ulated BM after 24-48hours in culture and M NCs and granulocytes were differentiated m orphologically. 150-300 metaphases were analysed for evidence o f donor sex chrom osom es.

4.3.2 E ngraftm ent levels and rates

This investigation revealed a clear difference in levels o f yc chain expressing donor cells, with norm al levels o f expression by T cells and significantly below normal levels expressed by B lymphocytes. O ther cases o f engraftm ent in the T lineage but failure o f donor cell repopulation o f the B lym phocytes or m onocyte lineages have been reported for XSCID as well as engraftm ent o f T eells but not granulocytes and B cells (Ginsburg et al., 1985; van Toi et al., 1998).

The results obtained by investigation o f patient D C ’s chim aerism levels at two time points touched upon the slow er engraftm ent rate o f B cells com pared to T cells. This is not unexpected from know ledge o f the strong selective grow th advantage o f yc chain expressing T cells over m utant non-expressing cells whereas early B cell precursors are subjected to no such com petitive effect (Puck et al., 1987; Conley et al., 1988). The different kinetics o f engraftm ent o f the T and B lineages has been confirm ed in a separate study w ith the m edian time for norm al function restoration following HLA- non-identical T cell depleted BM T o f 8.7 m onths for T cell and 14.9 m onths for B cells (Haddad et al., 1998).

Evidence suggests that the success o f transplantation and the likelihood o f graft versus host disease (G vH D ) are dependent upon the degree o f donor/recipient chimaerism within different sub-populations o f haem atopoietic cells. The absence o f T cell reconstitution at 6 m onths after H LA -non-identical T cell-depleted BM T was

dem onstrated to be associated w ith significantly low er survival rates, although there was no dependence on the level o f B cell chimaerism . In addition, a relationship between acute and chronic GvHD and slow er developm ent o f T cell function has been noted (H addad et al., 1998). H ow ever the full clinical relevance o f engraftm ent rates and levels has yet to be fully determined.

4.3.3 The functionality o f yc chain deficient B lym phocytes

An im portant issue to address is the determ ination o f the functional consequences o f partial or com plete absence o f donor B lym phocyte engraftm ent in these chimaeric patients. O f concern is the dependence on yc chain for class switching by autologous B cells even though donor yc chain expressing T cells are present. Evidence that yc chain deficient B lym phocytes are incapable o f class switching comes from X SCID carrier X- chrom osom e inactivation analysis by the separation o f surface IgM + and surface IgM- B cells prior to exposure to EBV and production o f somatic cell hybrids (Conley el a!.,

1988). Significant skewing was observed in the mature IgG and IgA producing surface IgM- B-lineage cells, whereas hybrids from the less mature IgM producing cells exhibited random X -chrom osom e inactivation. In addition, M atthews et al. (1995), dem onstrated that yc chain deficient B cells were unresponsive tow ards IL-2, a cytokine involved in the m aturation o f mature B cells to antibody producing plasm a cells. This is supported by analysis in my own laboratory, w hich indicates that following BM T treatment o f X SC ID patients, host yc chain deficient B lym phocytes are unable to class switch even after engraftm ent o f donor T cells (A Thrasher, personnel com munication). This finding stresses the im portance o f a BM T strategy optim ised for both T and B lymphocyte engraftm ent.