Reduced memory B-cell populations in boys with B-cell dysfunction after bone marrow transplantation for X-linked severe combined immunodeficiency

Authors


Associate Professor J. Ziegler, Department of Immunology/Allergy, Sydney Children's Hospital, High St., Randwick, NSW 2031, Australia. E-mail: J.Ziegler@unsw.edu.au

Abstract

X-linked severe combined immunodeficiency (XSCID) is a lethal disease resulting in death in infancy. In many instances, haploidentical bone marrow transplantation (BMT) offers reconstitution of T-cell immunity alone, with residual hypogammaglobulinaemia. The exact nature of B-cell dysfunction in these patients is unclear, although differentiation arrest of the B cells is a potential explanation. To ascertain the differentiation status of peripheral blood B lymphocytes from XSCID patients after BMT, the surface expression of CD19, CD10, CD34, CD5, serum immunoglogulin (sIg)M, sIgD, sIgG and CD27 on these B cells was investigated using three-colour flow cytometry. CD27 is a marker of memory B cells. Populations of CD19+IgMD B cells, CD19+IgM-only, CD19+IgG+CD27+ and CD19+IgM+ CD27+ B cells were found to be diminished in the XSCID patients after BMT with persistent hypogammaglobulinaemia, compared with both post-BMT patients with B-cell function and age-matched normal controls. This indicated the lack of CD19+IgMD B cells, which represent Ig isotype-switched B cells, as well as CD19+IgM-only and CD19+IgG+CD27+ or CD19+IgM+CD27+ memory B-cell populations. Interaction between CD27 and its ligand CD70 has been shown to induce IgG and IgM production by CD27+ B cells. Therefore, the lack of CD27/70 interaction is a probable explanation for the hypogammaglobulinaemia in these patients after BMT.

Severe combined immunodeficiency (SCID) is a group of genetic disorders characterized by profound impairment of both cellular and humoral immunity. Of these, the X-linked type (XSCID) caused by mutations in the interleukin 2 (IL-2) receptor γ chain gene (IL2RG) (Noguchi et al, 1993) is the most common (Puck et al, 1997; Ting et al, 1999). The IL-2Rγ chain has been renamed the common cytokine γ chain (γc), as it is shared by receptors for the cytokines IL-4, IL-7, IL-9 and IL-15. SCID can also be classified by immunological phenotype, in particular the T, B and natural killer (NK) cell populations. XSCID is characterized by profoundly diminished T and NK cell numbers with normal B-cell numbers, denoted as TB+NK. Atypical and less severe phenotypes of XSCID have also been reported (Schmalstieg et al, 1995; Sharfe et al, 1997). Other causes of TB+NK SCID include mutations in Janus kinase 3 (JAK3) (Russell et al, 1995), the primary intracellular signal transducer from γc, mutations in the IL-7Rα gene (Puel et al, 1998) and a small subset (5%) of unknown cause (Fischer et al, 1997).

Despite normal to increased B-cell numbers, children with XSCID universally suffer from hypogammaglobulinaemia pre- and, for many, post bone marrow transplantation (BMT) as well (Puck et al, 1997). There has been much debate over the mechanism of the B-cell dysfunction and it remains unresolved as to whether it is a result of lack of T-cell help or is a consequence of intrinsically defective B cells. The skewing of X chromosome inactivation involving B cells in heterozygous mothers strongly supports an intrinsic B-cell defect (Conley et al, 1988). The ability of XSCID B cells to proliferate and secrete immunoglobulin (Ig) in response to cytokines such as IL-2 and IL-15 has been shown to be abnormal or suboptimal, consistent with γc being a component of the IL-2 and IL-15 receptor complexes. However, activation of XSCID B cells induced by CD40L and IL-4 or IL-13, leading to upregulation of serum immunoglobulin (sIg)M, CD23 and Ig secretion, was similar to normal B cells, indicating that, with appropriate signalling, the B cells can function normally (Matthews et al, 1995).

As a result of their profound impairment of both cellular and humoral immunity, affected children with SCID usually present in the first few months of life with severe infections, persistent diarrhoea and failure to thrive. Until the recent report of successful clinical trials with gene therapy in XSCID (Cavazzana-Calvo et al, 2000), immune reconstitution by BMT was the only successful therapy and offered the only chance of survival (Report of a WHO scientific group, 1997). Histocompatible bone marrow transplant offers the best chance of complete immune reconstitution. However, matched sibling donors are usually not available. Haploidentical BMT with T-cell depletion has achieved survival rates of 52% to 78% in different studies (Fischer et al, 1990; Buckley et al, 1993, 1999). Despite donor T-cell engraftment and recovery of cellular immunity, humoral function is normal in only 50–60% of B+ SCID patients post BMT. This implies that, in a significant proportion of the post-BMT patients, the help from donor T cells is not sufficient for host B cells to function normally. However, this is not always the case. In a recent study, recovery of B-cell function in the absence of donor B cells but in the presence of engrafted donor T cells was found in γc-deficient patients following haploidentical BMT (Haddad et al, 1999). In these patients, host B cells were thought to be functional with the engrafted donor T cells. This suggested that T-cell help from the donor T cells may be adequate to restore function to the host γc-deficient B cells (Haddad et al, 1999). In contrast, another study found that in vivo B-cell function in γc-deficient patients after haploidentical BMT arose from donor B cells only. These donor B cells were present to different extents (White et al, 2000). The ongoing B-cell dysfunction and hypogammaglobulinaemia after BMT requires regular intravenous immunoglobulin (IVIG) infusions.

In a T cell-dependent immune response, naive B cells are unambiguously distinguished from antigen-experienced cells by expression of unmutated Ig variable (V) region genes. Following antigen activation, naive B cells are recruited into germinal centres (GCs). GCs are highly specialized areas of secondary lymphoid areas such as lymph nodes, spleen and tonsil (MacLennan et al, 1992; MacLennan, 1994). Within GCs, activated naïve B cells undergo proliferation, somatic hypermutation of Ig V region genes, Ig isotype switching and selection, after interaction with specific antigens (Liu et al, 1996a, b; Rajewsky, 1996). The process of somatic hypermutation generates a pool of high-affinity, antigen-specific B cells. High-affinity B cells can undergo further differentiation into either plasma cells or memory B cells (Rajewsky, 1996). A number of subsets of B cells with expression of different surface markers have recently been identified as memory cells. In addition to ‘classic’ class-switched memory B cells, populations of somatically mutated, IgM-expressing cells (IgM-only), IgD, as well as IgM+IgD+, cells that express somatically mutated Ig V region genes, were shown to co-express CD27 (Klein et al, 1997, 1998a). CD27 expression on B cells therefore correlates with presence of mutated V genes. Consequently, CD27 has been suggested to be a marker of memory B cells (Klein et al, 1998a; Tangye et al, 1998).

CD27 is a disulphide-linked homodimeric glycoprotein expressed on the majority of peripheral blood T lymphocytes and some B cells (Agematsu et al, 1997). The interaction between CD27 and its ligand, CD70 (Kobata et al, 1997), a molecule belonging to the tumour necrosis factor (TNF) receptor family found on peripheral T cells, has been demonstrated to drive B-cell IgG and IgM synthesis (Kobata et al, 1997). It is therefore a candidate for regulation of B-cell function in memory cells.

To determine the phenotype and state of differentiation of the dysfunctional B cells in XSCID patients post BMT, we have examined a cohort of patients requiring IVIG and compared them with XSCID patients with normal Ig levels after BMT and normal controls. As the contribution of host or donor B cells to in vivo B-cell function after BMT remains unresolved, if possible, analysis for chimaeric B cells was performed. We examined expression of CD19, CD10, CD34, IgM, IgD, CD5 and CD27, and have evidence that children with continuing B-cell dysfunction lack memory B cells.

Patients and methods

Patients XSCID patients with characterized γc mutations (Ting et al, 1999) were enrolled in this study and their characteristics are listed in Table I. Screening for γc mutations in these patients was achieved using single-strand conformation analysis (SSCP) with mutations confirmed by sequencing. Haploidentical BMT was performed at the Sydney Children's Hospital, Australia, from 1992 to 1996, with a follow-up period of 4–71/2 years. Two patients (5 and 6) with recovery of B-cell function after BMT had demonstrated normal responses to bacterial infections clinically, as well as normal serum Ig levels and production of specific antibodies to vaccines such as tetanus toxoid, diphtheria toxoid and Haemophilus influenza b (Hib). However, at last follow-up (4 years post BMT), patient 5, despite being clinically well with normal Ig levels, was found to have absent specific antibodies. He was given a booster vaccination of tetanus toxoid, diphtheria toxoid and Hib. An analysis of his B-cell phenotype from his peripheral blood before and after the booster vaccination was performed.

Table I.  Characteristics of BMT and post-BMT function in six patients with XSCID.

Patient

γc mutation
Result of
mutation
Current age
(years)
Age at BMT
(months)

CR
B-cell
chimaerism
IVIG
treatment
  1. BMT, bone marrow transplant; CR, chemotherapy regimen; IVIG, intravenous gammaglobulin; del, deletion; C, cytosine; T, Thymine; G, guanine; N, normal; Cy, cyclophosphamide; ATG, anti-thymocyte globulin; Mel, melphalan; TT, thiotepa, ND, not done; H, host.

1C→T cDNA684 (Exon 5)Arg. To Trp.5·258Nil94% HYes
2G→T cDNA868 (Exon 6)Arg. To Leu.6·336Nil94% HYes
3A insert cDNA738 (Exon 5)Premature stop codon7·582Nil95% HYes
4A→G cDNA15 (Exon 1)Met. To val.4·426Cy, ATG, TTNDYes
5G del. cDNA736 (Exon 5)Premature stop codon5·337Nil71% HNo
6C→T cDNA678 (Exon 5)Arg. To cys.7·751Cy, Mel, ATGNDNo

BMT characteristics BMT was performed from the ages 1–8 months (mean 5 months), as summarized in Table I. In each case, haploidentical marrow was obtained from a parental donor. T-cell depletion was achieved using soybean lectin and sheep erythrocyte agglutination (Reisner et al, 1983). Two patients had a combination of cyclophosphamide (Cy) 50 mg/kg (Astra Medica), melphalan (Mel) 6·7 mg/kg (Glaxo Wellcome) and anti-thymocyte globulin (ATG) 12 mg/kg (Pharmacia and Upjohn) or thiotepa (TT) 5 mg/kg (Lederle) prior to BMT, while the other four patients had no chemotherapy.

B-cell function Serum Ig levels were measured using nephelometry. Specific antibodies to tetanus, diphtheria and Hib were measured using enzyme-linked immunosorbent assay (ELISA) at the Immunology Laboratory at the New Children's Hospital, Sydney (Farzad et al, 1986; Johnson et al, 1996).

Controls Blood was obtained with informed consent from normal control children from aged 23 months to 7 years. Peripheral blood mononuclear cells were isolated and cryopreserved at −70°C.

Blood collection Peripheral blood samples were collected and lymphocyte separation was performed within 24 h.

B-cell chimaerism Patients who received donor maternal marrow had B-cell chimaerism studies performed using fluorescent in situ hybridization (FISH) using X and Y chromosome probes.

Cell isolation/preparation/fixation Peripheral blood monoclear cells were separated from heparin-treated blood using Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. Isolated cells were frozen in 10% dimethyl sulphoxide (DMSO) and stored in liquid nitrogen at 5 × 106cells/ml if not used immediately.

Antibody/reagents Monoclonal antibodies (mAbs) used in this study were phycoerythrin (PE)-labelled anti-IgM, anti-IgG, anti-CD5, anti-CD80, anti-CD34, fluorescein isothiocyanate (FITC)-labelled anti-IgD, anti-IgM, anti-CD27, anti-CD10 and peridinin chlorophyll (PerCp)-labelled anti-CD19. Fluorochrome-conjugated mouse IgG mAbs were used as isotype controls. All of the above mAbs were purchased from Becton Dickinson/Pharmingen, Sydney, Australia.

Immunofluorescent staining and FACS analysis Peripheral blood mononuclear cells were incubated with 10 μl of each appropriate mAb at 4°C for 30 min, followed by two washes in FACS buffer (phosphate-buffered saline, 0·4% fetal calf serum, 0·2% sodium azide) and, finally, fixation in 1% paraformaldehyde. Analysis of fixed cells was carried out on a FACScan flow cytometer (Becton Dickinson). Between 10 × 104 and 50 × 104 events were collected per sample and the data were analysed using Cell Quest software (Becton Dickinson).

Statistical analysis Statistical analysis was performed using Statview 5·0 software (SAS Institute, Cary, NC, USA). Intergroup comparisons were carried out using the non-parametric Mann–Whitney U-test.

Results

The XSCID patients and their corresponding γc mutations (Ting et al, 1999) are summarized in Table I. Each received a T cell-depleted, haploidentical BMT from a parent. Two patients had pre-BMT chemotherapy regimens (CRs) of Cy, ATG, TT (patient 3) and Cy, Mel, ALG (patient 6). All six patients had normal T-cell numbers and clinically normal function after BMT. Four patients had B-cell dysfunction post BMT, indicated by serum hypogammaglobulinaemia and the continuing need for regular IVIG. The two patients with normal serum Ig levels had demonstrated normal production of specific antibodies to tetanus, diphtheria and Hib.

Absent IgMIgDCD19+ B population in XSCID patients with B-cell dysfunction after BMT

A distinct population of IgMIgDCD19+ B cells, which comprise 16% (range 7–25%) of CD19+ B cells, was consistently detected in the peripheral blood of normal controls (Fig 1C, Table II). These probably represent Ig class-switched B cells. This population of B cells was markedly diminished in the XSCID patients with B-cell dysfunction (Fig 1A) (mean 0%, range 0–2%, Cohort A, Table II). Three percent of CD19+ B cells from patient 6 who had B-cell function after BMT were IgMD (Table II). Interestingly, the proportion of IgMD19+ B cells in the peripheral blood of patient 5 increased from 1·3% (Fig. 2A) to 4·5% (Fig. 2B) 10 d after the booster vaccination. Overall, there was a significantly reduced (P = 0·01 by Mann–Whitney) percentage of IgMIgD19+ B cells in the XSCID patients with B-cell dysfunction compared with the normals.

Figure 1.

Phenotypic analysis of peripheral blood B lymphocytes. Expression of IgM and IgD on CD19+ B cells. Mononuclear cells from the peripheral blood of three different cohorts were incubated with PerCP-labelled anti-CD19, PE-labelled IgM and FITC-labelled IgD. CD19+ cells were gated and the fluorescence of each population was assessed using three-colour flow cytometric analysis. Representative results from (A) XSCID post BMT with hypogammaglobulinemia, (B) XSCID post BMT with B-cell function and (C) a normal control are shown. IgMD CD19+ B cells represent switched memory B cells.

Table II.  Summary of percentages of subpopulations of CD19+ B cells in the subjects from cohort A (XSCID post BMT + hypogammaglobulinaemia), Cohort B (XSCID post BMT + B-cell function) and Cohort C (age-matched controls).


Patient
% of CD19+ B cells
IgM+D+IgM+DIgMDIgMD+IgG+27+IgM+27+
Cohort A
 178122000
 29510401
 3973001
 496003
Mean9210701
Range78–960–30–20–2000–1
Cohort B
 582557313
 69153338
Mean87545311
Controls
 N1498251832
 N27951514
 N373219633
 N48557327
 N5767126410
 N673219633
Mean73516735
Range49–852–87–251–182–42–10
P-value A to C (Mann–Whitney)0·030·030·010·670·050·02
Figure 2.

Comparison of phenotypic analyses of peripheral blood B cells from an XSCID post-BMT patient (A and B) before and (C and D) after booster immunization. Peripheral monocytes were stained for PerCP-anti-CD19, PE-conjugated anti-IgM and FITC-conjugated anti-CD27 for three-colour flow cytometric analysis.

CD27+CD19+ B cells

CD27 has been described as a marker for human memory B cells (Kondo et al, 1994; Klein et al, 1998a; Tangye et al, 1998). We therefore estimated the proportions of memory B cells in the peripheral blood of XSCID patients post BMT by staining for CD27. In normal healthy donors, a mean of 8% (range 4–14%) of CD19+ B cells expressed CD27 [ Fig 3C, (i) and (ii)]. Consistent with the above result, 14% (range 11–16%) of B cells from XSCID post BMT with B-cell function stained positively for CD27 [Fig 3B, (i) and (ii)] (Table II). However, there was no significant staining for CD27 in the CD19+ B-cell population in patients with B-cell dysfunction [Fig 3A, (i) and (ii)] and the differences in IgG+CD27+ and the IgM+CD27+ subpopulations were significantly different from controls (P = 0·05 and 0·02 respectively).

Figure 3.

Fluorescence analysis for CD27+ memory B cells. Peripheral blood mononuclear cells from XSCID patients post BMT (A) with B-cell dysfunction, (B) without B-cell dysfunction and (C) a normal control were incubated with CD19-perCP, CD27-FITC and PE-IgG or IgM. CD19+B cells were gated for analysis of IgG or IgM expression with CD27.

Diminished IgM-only memory B cells

Figure 1A also shows an almost complete absence of another B-cell population in patients with XSCID post-BMT B-cell dysfunction, the IgM-only CD19+ B cells which belong to another subset of memory B cells (Klein et al, 1998b). This population represents 7% of CD19+ B cells in the normal controls (Fig 1C) (P = 0·03) and represents the somatically mutated, IgM-expressing memory B cells.

Absent CD5, CD34, CD10 expression

CD5+ B cells have been reported to be one of the first cell populations to appear post BMT (Antin et al, 1987). They are also thought to belong to a separate B-cell lineage and do not regularly participate in T cell-dependent immune responses (Kipps, 1989; Linton et al, 1992; Stall et al, 1996). Examination of cells for CD5 expression was carried out to ascertain the lineage of the populations seen. CD34 and CD10, surface markers of early developmental stages of B cells and their progenitors, are expressed up to pro/pre-B and immature B stages of differentiation (Ghia et al, 1996). In contrast, one of the earliest expressed surface markers of B-lineage cells is CD19, which is expressed throughout B-cell development, except in plasma cells. Staining for these markers of early developmental stages was performed to look for possible arrest in B-cell development. There was no significant staining for CD34 or CD10 when co-stained with IgM and gated on CD19+ B cells (data not shown). Staining for CD5 was also negative, indicating these B cells were not of B-1 lineage.

Chimaerism studies

Analysis for chimaeric donor and host B cells using X and Y probes in FISH studies was only informative for patients with female donors. As the analysis was performed with Epstein-Barr virus (EBV)-transformed B cells, this study was qualitative owing to possible clonal selection during the B-cell transformation. Only one (patient 5) of the four patients tested had normal clinical status. Donor cells were present in all four patients, although patient 5 had a much higher percentage of donor cells, 29% compared with < 5% in the remainder (column 7 in Table I).

Patient 5

It was interesting to note that patient 5 had no antibodies to tetanus, diphtheria and Hib, even though the previous blood test a year prior showed these to be present. Booster immunization to tetanus, diphtheria and Hib was given and, 10 d later, high to moderately high levels of these specific antibodies (tetanus Ab 5·89 EU/ml, diphtheria Ab 0·24 EU/ml, Hib Ab > 10 μg/ml) were detected in the peripheral blood, which was consistent with a strong secondary response. When repeated 8 months later, the tetanus and Hib AB levels were 3·07 EU/ml and 1·93 μg/ml respectively. We analysed the patient's B-cell phenotype before (Fig 2A and B) and 10 d after (Fig 2C and D) the booster vaccination. The populations of IgMDCD19+ (Fig 2C) (switched B cells) and CD27+ memory B cells were clearly present in the post-booster peripheral blood analysis only (Fig 2C and D).

Discussion

We report in this study the absence of memory B cells in XSCID post-BMT patients with B-cell dysfunction as a probable explanation for their ongoing hypogammaglobulinaemia. Although numerous studies have confirmed the large proportion of patients with ongoing B-cell dysfunction following a haploidentical BMT (40–50%) (Buckley et al, 1999; Haddad et al, 1999), only a few have phenotyped the peripheral B cells in these patients. Small et al (1989) demonstrated that SCID B cells had surface markers similar to cord blood B cells, such as CD1c, CD38 and CD23, and that expression of these antigens decreased with age. A recent study of long-term chimaerism and B-cell function after BMT in B+ SCID in 22 patients found four patients with B cells of host origin, normal B-cell function and host memory B cells (Haddad et al, 1999). We have extended this study to include surface markers crucial for differentiation status and identification of memory B cells such as IgM, IgD and IgG, in addition to markers of less mature B cells such as CD10 and CD34. It was important to include CD5 to ascertain whether the cells were of B-1 or B-2 lineage.

We found that a clear distinction between the CD19+ B cells from the post-BMT XSCID patients with B-cell dysfunction and those of the controls was the near absence of the IgMIgD population in the former group. This probably represents Ig isotype-switched B cells as the cells lacked expression of CD34 or CD10, implying that the differentiation state of these cells did not correspond to the pre-B or immature B-cell stage. This population of B cells is clearly present in patients with B-cell function post BMT. Thus, analysis of the IgMIgD19+ B-cell population may provide a useful tool for monitoring B-cell recovery following BMT, as the level of serum Ig may be difficult to interpret while the patient is on replacement IVIG.

The IgM-only B cells were also found to be reduced in patients lacking B-cell immunity post BMT compared with normals. In peripheral blood, spleen and bone marrow, memory B cells have been identified that expresss either class-switched Ig isotypes, IgM and IgD, or IgM only (Klein et al, 1997, 1998a; Paramithiotis & Cooper, 1997; Tangye et al, 1998). This observation of a diminished IgM-only memory B-cell population in this cohort of hypogammaglobulinaemic XSCID post-BMT patients supports our findings of an absence of all subsets of memory B cells. Absence of CD5 expression indicates that the subpopulations of IgM-D-CD19+, IgM-alone and CD27+ B cells were not of the B-1 lineage.

The presence of CD27 in different subpopulations of memory B cells resulted in the suggestion that CD27 is a marker of memory B cells in human peripheral blood (Klein et al, 1998a) and spleen (Tangye et al, 1998). As with a previous study (Haddad et al, 1999), we confirmed the absence of CD27+ memory B cells in the patients with B-cell dysfunction. However, we further defined this population to include both unswitched (IgM+D+) and isotype-switched (IgG+) B cells for the co-expression of CD27. The expression of CD27 on IgMD19+ B cells in normals and XSCID patients with B-cell function after BMT, but not in those patients without B-cell function, is consistent with previous studies demonstrating that CD27 is expressed on memory B cells. This confirmation is important as there are a number of subpopulations of memory B cells identified by various combinations of surface Ig expression so that a marker that positively identifies the memory B-cell population would be very useful for other studies involving B-cell subsets. CD27 has been suggested to have a role in regulation of B-cell function and plasma cell development. Absence of CD27 would result in an impaired CD27/70 interaction, which has been reported to drive CD27+ B cells to IgG and IgM production (Kobata et al, 1997) and would probably have profound implications for the persistent hypogammaglobulinemia in patients with XSCID post BMT. IgDCD27+ memory B cells were found to be greatly reduced in another X-linked immunodeficiency, the hyperIgM syndrome, which was postulated to be the reason for the low IgG production in this disorder (Agematsu et al, 1998). In normal healthy individuals, activation of B cells increases CD27 expression, although chronic stimulation also leads to the disappearance of CD27. Further studies to investigate the reason for the lack of CD27 and examine the means of stimulating CD27 expression may well lead to successful alternate therapies for the persistent humoral dysfunction other than regular IVIG.

Patient 5 provided an excellent opportunity to examine the profile of subpopulations of B cells in a patient with B-cell immunity before and after a booster vaccination. It was interesting to note the resurgence of the IgMIgDCD19+ B cells, as well as CD27+ IgG and IgM during the antibody response (7–10 d) after the booster vaccination. One possible explanation is the mobilization of memory B cells from other organs such as the spleen or the tonsils into peripheral blood. A further implication from this finding is that memory B cells can be produced following antigen challenge in these patients. The Ig response to the tetanus toxoid had persisted on retesting 8 months after the toxoid administration. It remains possible that the memory B cells in patient 5 may not have long-term survival compared with memory B cells in normals. That being the case, interpretation of protective levels of specific antibodies may become less clear and possibly vary depending on time course following vaccination. Further studies of B-cell characterization with different scenarios, for example, B-cell phenotypes at different time-points following vaccination in patients with various immune-deficient states, will need to be performed to clarify this issue. The chimaerism studies revealed the higher percentage of donor B cells present in patient 5. Although the presence of these donor B cells may explain the recovery of this patient's B-cell function, it would not explain the lack of B-cell function in the other three patients tested who also had donor B cells present, unless the presence of a critical level of B cells is required to provide function. Although known to cause graft rejection in BMT (Shlomchik et al, 1999), the role of antigen-presenting cells (APCs) in the recovery of B-cell function in XSCID following BMT is unclear. Three of the four γc-deficient patients with host B cells after an HLA-identical BMT had B-cell function compared with only one of the 11 γc-deficient patients with host B cells after HLA non-identical BMT, suggesting an important role for HLA matching. With donor T cells being present in both groups, a remaining important population of cells to be considered in the B-cell response to antigens is the APCs. While it is possible that HLA disparity between donor APCs and host B cells may result in problems with host B-cell maturation and responsiveness, HLA matching of donor APCs and engrafted donor B cells has not restored B-cell function in all cases. The APCs role in antigen presentation to T cells, which are donor in origin, appears normal. Therefore, XSCID patients provide great opportunities for further studies into the role of HLA and T–B cooperation following antigen presentation.

XSCID has also provided an excellent system for the study and characterization of B-cell maturation and differentiation. The findings in this study of absent IgMIgDCD19+ B cells, IgM-only cells, as well as CD27+ memory B cells, in the XSCID with B-cell dysfunction group would suggest that their B cells were able to undergo normal maturation at least to the stage of mature B cells. In those with B-cell dysfunction post BMT, in whom the majority of B cells are of host origin, it appears that these B cells were unable to further differentiate to memory B cells. It would be of great interest to extend this study to investigate various factors such as stimulation by cytokines to encourage differentiation into memory B cells and Ig production as a possible alternate mode of therapy to treat the ongoing B-cell dysfunction. The use of surface expression of markers such as IgM, IgD, CD27 and IgG has been shown to be useful for studying the phenotype and differentiation status of B cells (Tangye et al, 1998). The same can be applied to study other poorly understood immune dysfunctions in both congenital and acquired conditions, as well as for monitoring memory B-cell responses in vaccine trials and immunodeficient patients. An important clinical implication from the findings in this study is that greater caution needs to be exercised in those with absent memory B cells. The reason is that their ability to maintain specific antibody production remains unclear, therefore more frequent and better timed testing for the specific antibody levels may assist in the management of these patients.

Acknowledgments

S. S. Ting is supported by the NH & MRC medical postgraduate research scholarship 987606 and S. G. Tangye is supported by a U 2000 post-doctoral research fellowship and grant awarded by the University of Sydney, Sydney, Australia.

We are grateful to the patients and their doctors for assistance with blood samples. We especially thank Angel Jaramillo (Paediatric Research Laboratory, Sydney Children's Hospital) for his invaluable assistance and Alex Kovacic (Cytogenetics Department, Prince of Wales Hospital, Sydney) for performing the FISH studies.

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